KR102081657B1 - Humanized light chain mice - Google Patents

Abstract

Non-human animals, tissues, cells, and genetic material are provided that include ADAM6 active functionality in mice and also include modifications of endogenous non-human heavy chain immunoglobulin sequences, wherein the non-human animal is a human immunoglobulin heavy chain variable. Domain and cognate human immunoglobulin λ light chain variable domain.

Description

Humanized light chain mouse {HUMANIZED LIGHT CHAIN MICE}

Genetically modified non-human fertile animals express human immunoglobulin λ light chain variable sequences that are cognate with human heavy chain variable sequences. Genetically modified mice, cells, embryos and tissues comprising a nucleic acid sequence encoding ADAM6 functionality within the mouse ADAM6 site are described, wherein the mice, cells, embryos and tissues are regenerated to form functional immunoglobulin light chain variable domains. Contains human immunoglobulin lambda light chain gene segments that can be arranged. Modifications include human and/or humanized immunoglobulin sites. Mice containing ADAM6 function, including mice containing an ectopic nucleic acid sequence encoding the ADAM6 protein, have been described. Genetically modified male mice comprising genetic modification of the gene locus of the _{endogenous mouse immunoglobulin V H} region, and further comprising ADAM6 activity, including mice comprising an ectopic nucleic acid sequence that restores fertility to male mice are described. Has been.

Including deletion or modification of the endogenous ADAM6 gene or its homolog or ortholog, and including genetic modifications that restore ADAM6 (or its homolog or ortholog), Genetically modified non-human fertile animals function as a whole or in part, wherein the non-human animal expresses a human immunoglobulin λ variable sequence in the context of a λ or κ light chain constant sequence.

In the past 20 years, pharmaceutical applications for antibodies have been devoted to the manufacture of antibodies suitable for use as human therapeutics. Early antibody therapeutics based on mouse antibodies were not ideal as human therapeutics because repeated administration of mouse antibodies to humans leads to immunogenicity problems that may prove wrong with long-term treatment regimens. Solutions based on humanization of mouse antibodies have been developed to make them look more human-like or less mouse-like. Methods for expressing human immunoglobulin sequences for use in antibodies were, as a result, mostly based on in vitro expression of human immunoglobulin libraries in phage, bacteria or yeast. Finally, in transchromosomal or transgenic mice in which the endogenous immunoglobulin locus has been disabled, and in mice injected with human hematopoietic cells, an attempt was made to construct useful human antibodies from human lymphocytes in vitro. . In these transgenic mice, it was necessary to disable the endogenous mouse immunoglobulin gene so that the complete human transgene, which was randomly incorporated, functions as a source of the immunoglobulin sequence expressed in the mouse. Such mice can make human antibodies suitable for use as human therapeutics, but these mice show substantial problems with their immune system. These problems (1) make it impossible for mice to generate a sufficiently diverse antibody repertoire, (2) require the use of a wide range of remanufactured solutions, and (3) possibly the next best clone due to incompatibility between human and mouse elements. There is a likelihood of providing a selection process, and (4) giving these mice an unreliable source of a vast and diverse population of human variable sequences that are required to be truly useful in making human therapeutics.

Transgenic mice containing the intact human antibody transgene are unrearranged human immunoglobulin heavy chain variable sequences (V, D and J sequences) linked to human heavy chain constant sequences and unrearranged linked to human light chain constant sequences. It includes randomly inserted transgenes comprising human immunoglobulin light chain variable sequences (V and J). Thus, the mouse produces a rearranged antibody gene from a site other than the endogenous mouse site, wherein the rearranged antibody gene is completely human. In general, mice with at least some human λ sequences have also been reported, but mice comprise a human heavy chain sequence and a human κ light chain sequence. Transgenic mice generally have damaged and non-functional endogenous immunoglobulin sites, or knockout of endogenous immunoglobulin sites, making it impossible to rearrange human antibody sequences at endogenous mouse immunoglobulin sites. These unexpected changes in the transgenic mice are probably due, at least in part, to the suboptimal clonal selection process that connects with intact human antibody molecules within the endogenous mouse selection system, making them less likely to generate a sufficiently diverse human antibody repertoire in mice. Make it optimal.

There remains a need in the art to make improved genetically modified non-human animals useful for generating immunoglobulin sequences, including human antibody sequences, and also useful for generating a sufficiently diverse human antibody repertoire. In addition, proteins can be constructed from altered immunoglobulin sites, including sites containing a sufficient variety of human λ and/or human κ light chain variable sequences, or include human heavy chain variable domains cognate with human λ or human κ variable domains. There remains a need for mice that can rearrange immunoglobulin gene segments to form useful rearranged immunoglobulin genes. There is also a need for non-human animals capable of generating antibody variable regions from both human κ and human λ segments, where the human κ and human λ segments are cognate with the human heavy chain variable domain. There is also a need for an increased use of human λ sequences in genetically modified animals.

A genetically modified non-human animal is described that contains a modification that reduces or eliminates the activity of the ADAM6 gene or its homologues or orthologs, wherein the modification results in a loss of fertility and the animal is lost or reduced. A sequence encoding an activity that complements or restores ADAM6 activity (or homolog or ortholog activity), and non-human animals allow them to be cognate with the human immunoglobulin λ light chain variable region. It further includes a modification that allows the region to be expressed. In various aspects, the human immunoglobulin λ light chain variable region is expressed by fusion to the λ or κ constant region.

In various aspects, the sequence encoding ADAM6 activity is contiguous with the human immunoglobulin sequence. In various aspects, the sequence encoding ADAM6 activity is contiguous with the non-human immunoglobulin sequence. In various aspects, the sequence is on the same chromosome as the endogenous non-human immunoglobulin heavy chain site of the non-human animal. In various aspects, the sequence is on a different chromosome than the immunoglobulin heavy chain site of the non-human animal.

A genetically modified non-human animal comprising a modification that retains the activity of the ADAM6 gene or its homolog or ortholog is described, wherein the modification is one or more human immunity upstream of the non-human immunoglobulin heavy chain constant region. It comprises an insert of a globulin heavy chain gene segment, wherein the non-human animal further comprises a modification capable of causing them to express a human immunoglobulin λ light chain variable region that is cognate with the human immunoglobulin heavy chain variable region. In various aspects, the human immunoglobulin λ light chain variable region is expressed by fusion to the λ or κ constant region.

In various aspects, the insertion of one or more human immunoglobulin heavy chain gene segments is performed downstream or 3′ of the ADAM6 gene of a non-human animal. In various aspects, the insertion of one or more human immunoglobulin heavy chain gene segments is such that the ADAM6 activity of the non-human animal is at the same or comparable level as the non-human animal that does not contain such insertion. It is performed in such a way that the gene(s) are not destroyed, deleted and/or functionally silent. Exemplary disruption, deletion and/or functional silencing modifications include any modification that results in a reduction, elimination and/or loss of the activity of ADAM6 protein(s) encoded by ADAM6 gene(s) in non-human animals. .

In one aspect, a nucleic acid construct, a cell, an embryo, a mouse and a method for constructing a mouse comprising a non-functional endogenous mouse ADAM6 protein or a modification resulting in the ADAM6 gene (e.g., knockout or deletion of the endogenous ADAM6 gene) Is provided, wherein the mouse comprises a nucleic acid sequence encoding a functional ADAM6 protein or an ortholog or homolog or fragment thereof in a male mouse.

In one aspect, a nucleic acid construct, cell, embryo, mouse and method are provided for constructing a mouse comprising a modification of an endogenous mouse immunoglobulin site, wherein the mouse is a functional ADAM6 protein or an ortholog thereof in a male mouse. Or a homologue or fragment. In one embodiment, the endogenous mouse immunoglobulin site is an immunoglobulin heavy chain site, and the modification reduces or eliminates ADAM6 activity in cells or tissues of a male mouse.

In one aspect, there is provided a mouse comprising an ectopic nucleotide sequence encoding mouse ADAM6 or an ortholog or homolog or functional fragment thereof; Also provided are mice comprising an endogenous nucleotide sequence encoding mouse ADAM6 or an ortholog or homolog or fragment thereof and comprising at least one genetic modification of a heavy chain immunoglobulin site.

In one aspect, a method for constructing a mouse comprising modification of an endogenous mouse immunoglobulin site is provided, wherein the mouse comprises a functional ADAM6 protein or an ortholog or homolog or fragment thereof in a male mouse.

In one aspect, a method for constructing a mouse comprising genetic modification of a heavy chain immunoglobulin site is provided, wherein application of this method results in a male mouse comprising a modified heavy chain immunoglobulin site (or deletion thereof), and , The male mouse can produce offspring by mating. In one embodiment, the male mouse is capable of producing sperm that can move through the mouse fallopian tube from the mouse uterus to fertilize the mouse egg.

In one aspect, a method for constructing a mouse comprising genetic modification of an immunoglobulin heavy chain locus and an immunoglobulin light chain locus is provided, wherein the application of this method to modify the heavy chain locus is a male mouse showing a decrease in fertility. Occurs in, and mice contain genetic modifications that fully or partially restore the decline in fertility. In various embodiments, the reduction in fertility is characterized by the inability to move the sperm of the male mouse through the mouse fallopian tube from the mouse uterus to fertilize the mouse egg. In various embodiments, the decrease in fertility is characterized by sperm that exhibit immobilization defects in vivo. In various embodiments, the genetic modification that fully or partially restores the decrease in fertility is a nucleic acid sequence encoding a functional mouse ADAM6 gene or an ortholog or homolog or fragment thereof in a male mouse.

In one embodiment, the genetic modification comprises replacing an endogenous immunoglobulin heavy chain variable site with an immunoglobulin heavy chain variable site of another species (eg, a non-mouse species). In one embodiment, the genetic modification comprises the insertion of an orthologous (ie, orthologous) immunoglobulin heavy chain variable site into an endogenous immunoglobulin heavy chain variable site. In a specific embodiment, the species is human. In one embodiment, the genetic modification comprises a deletion in whole or in part of the endogenous immunoglobulin heavy chain variable locus, wherein the deletion results in a loss of endogenous ADAM6 function. In a specific embodiment, the loss of endogenous ADAM6 function is associated with a decrease in fertility in male mice.

In one embodiment, the genetic modification comprises full or partial inactivation of the endogenous non-human immunoglobulin heavy chain variable locus, wherein the inactivation does not result in a loss of endogenous ADAM6 function. Inactivation is the replacement of one or more endogenous non-human gene segments resulting in an endogenous non-human immunoglobulin heavy chain locus making it substantially impossible to rearrange to encode a heavy chain of an antibody comprising an endogenous non-human gene segment, or May contain fruit Inactivation may include other modifications that make it impossible to rearrange to encode the heavy chain of the antibody to the endogenous immunoglobulin heavy chain site, where the modification does not involve replacement or deletion of an endogenous gene segment. Exemplary modifications include chromosomal inversion and/or translocation mediated by molecular techniques, such as molecular techniques (eg, Cre-lox techniques) that use precise placement of site-specific recombination sites. Other exemplary modifications include disabling an operative linkage between a non-human immunoglobulin variable gene segment and a non-human immunoglobulin constant region.

_{In one embodiment, the genetic modification is one or more human V H} gene segments of another species (e.g., non-mouse species) operably linked to one or more constant region sequences (e.g., IgM and/or IgG genes), 1 And inserting DNA fragments comprising at least one human D _H gene segment and at least one human J _H gene segment into the genome of a non-human animal. In one embodiment, the DNA fragment can be rearranged in the genome of a non-human animal to form a sequence encoding the heavy chain variable domain of the antibody. In one embodiment, the species is human. In one embodiment, the genetic modification is a down of the endogenous ADAM6 gene of a non-human animal such that ADAM6 activity (such as expression and/or function of the encoded protein) is identical or comparable to that of a non-human animal without insert. Stream or 3′ of one or more human immunoglobulin heavy chain gene segments.

In one aspect, due to the reduction or elimination of endogenous ADAM6 function, male mice with the deformity have reduced fertility (e.g., a highly reduced ability to produce offspring by mating), or endogenous to be inherently infertile. A mouse is provided comprising a modification that reduces or eliminates mouse ADAM6 expression from an ADAM6 allele, wherein the mouse further comprises an ectopic ADAM6 sequence or a homolog or ortholog or functional fragment thereof. In one aspect, the modification that reduces or eliminates mouse ADAM6 expression is a modification of the mouse immunoglobulin site (eg, insertion, deletion, replacement, etc.).

In one embodiment, the reduction or loss of ADAM6 function comprises the inability or substantially inability of the mouse to produce sperm that can move through the mouse oviduct from the mouse uterus to fertilize the mouse egg. In a specific embodiment, at least about 95%, 96%, 97%, 98% or 99% of the sperm cells produced in the ejaculation volume of the mouse are unable to fertilize the mouse egg by moving through the fallopian tube in vivo after mating. Let's do it.

In one embodiment, the reduction or loss of ADAM6 function comprises the inability to form or a substantial inability to form a complex of ADAM2 and/or ADAM3 and/or ADAM6 on the surface of a sperm cell of a mouse. In one embodiment, the loss of ADAM6 function comprises a substantial inability to fertilize a mouse ovum by mating with a female mouse.

In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 gene and comprises a protein (or an ectopic nucleotide sequence encoding the protein) that confers ADAM6 functionality to the mouse. In one embodiment, the mouse is a male mouse, and the functionality includes increased fertility compared to mice lacking the functional endogenous ADAM6 gene.

In one embodiment, the protein is encoded by a genomic sequence located within an immunoglobulin site in the germline of a mouse. In a specific embodiment, the immunoglobulin site is a heavy chain site. In another specific embodiment, the heavy chain locus comprises at least one human V _H , at least one human D _H and at least one human J _H gene segment. In one embodiment, the ectopic protein is encoded by a genomic sequence located within a non-immunoglobulin site in the live gland of a mouse. In one embodiment, the non-immunoglobulin site is a transcriptionally active site. In a specific embodiment, the transcriptional active site is the ROSA26 site. In a specific embodiment, the site of transcriptional activity is associated with tissue-specific expression. In one embodiment, tissue-specific expression is present in regenerated tissue. In one embodiment, the protein is encoded by a genomic sequence randomly inserted into the germline of the mouse.

In one embodiment, the mouse comprises a human or chimeric human/mouse or chimeric human/rat light chain (eg, human variable, mouse or rat constant) and a chimeric human variable/mouse or rat constant heavy chain. In a specific embodiment, the mouse comprises a transgene operably linked to a transcriptionally active promoter, such as a chimeric human variable/rat or mouse constant light chain gene operably linked to the ROSA26 promoter. In another specific embodiment, the chimeric human/mouse or rat light chain transgene comprises a rearranged human light chain variable region sequence within the germline of a mouse.

In one embodiment, the ectopic nucleotide sequence is located within an immunoglobulin site in the germline of a mouse. In a specific embodiment, the immunoglobulin site is a heavy chain site. In one embodiment, the heavy chain locus comprises at least one human V _H , at least one human D _H and at least one human J _H gene segment. In one embodiment, the ectopic nucleotide sequence is located within a non-immunoglobulin site in the germline of a mouse. In one embodiment, the non-immunoglobulin site is a transcriptionally active site. In a specific embodiment, the transcriptional active site is the ROSA26 site. In one embodiment, the ectopic nucleotide sequence is randomly inserted and positioned into the germline of the mouse.

In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 gene, wherein the mouse comprises an ectopic nucleotide sequence that compensates for the loss of mouse ADAM6 function. In one embodiment, the ectopic nucleotide sequence confers to the mouse the ability to produce progeny comparable to a corresponding wild-type mouse comprising the functional endogenous ADAM6 gene. In one embodiment, such sequences confer to the mouse the ability to form a complex of ADAM2 and/or ADAM3 and/or ADAM6 on the surface of the mouse sperm cells. In one embodiment, the sequence confers to the mouse the ability to move through the mouse fallopian tube from the mouse uterus to fertilize the egg.

In one embodiment, mice lacking a functional endogenous ADAM6 gene and comprising an ectopic nucleotide sequence produce at least about 50%, 60%, 70%, 80% or 90% of the number of litters of wild-type mice of the same age. And strains are produced in a time period of 6 months.

In one embodiment, mice lacking a functional endogenous ADAM6 gene and comprising an ectopic nucleotide sequence are mated over substantially the same time period and under substantially the same conditions lacking a functional endogenous ADAM6 gene and lacking an ectopic nucleotide sequence. At least about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 4 times, about 6 times, about 7 times, about when mated over a 6-month time period than mice of the same age and the same or similar strains. It produces 8 times or about 10 times or more progeny.

In one embodiment, a mouse lacking a functional endogenous ADAM6 gene and comprising an ectopic nucleotide sequence lacks the functional endogenous ADAM6 gene and lacks an ectopic nucleotide sequence, and a 4- or 6-month crossing than mice mated in the same time period. On average, at least about 2, 3, or 4 times more pups are produced per litter during the period.

In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence is a male mouse, and the male mouse lacks the functional endogenous ADAM6 gene and is ectopic when recovered from the fallopian tube at about 5 to 6 hours after mating. At least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, 100 times, 110 times or 120 times than mice lacking nucleotide sequence Produces sperm that reflects the movement of the fallopian tubes or more.

In one embodiment, a mouse lacking a functional endogenous ADAM6 gene and comprising an ectopic nucleotide sequence is capable of crossing the uterus and entering the fallopian tube within about 6 hours at approximately the same efficiency as sperm from a wild-type mouse when mated with a female mouse. Produce sperm that is present.

In one embodiment, a mouse lacking the functional endogenous ADAM6 gene and comprising an ectopic nucleotide sequence is about 1.5 times, about 2 times, about 1.5 times, about 2 times, about a time period comparable to a mouse lacking the functional endogenous ADAM6 gene and lacking the ectopic nucleotide sequence. Produces three times or about four times or more litter.

In one aspect, there is provided a mouse comprising a non-mouse nucleic acid sequence encoding an immunoglobulin protein, wherein the non-mouse immunoglobulin sequence comprises a mouse ADAM6 gene or a homologue thereof or an insert of an ortholog or functional fragment. do. In one embodiment, the non-mouse immunoglobulin sequence comprises a human immunoglobulin sequence. In one embodiment, the sequence comprises a human immunoglobulin heavy chain sequence. In one embodiment, the sequence comprises a human immunoglobulin light chain sequence. In one embodiment, the sequence comprises at least one V gene segment, at least one D gene segment, and at least one J gene segment; In one embodiment, the sequence comprises at least one V gene segment and at least one J gene segment. In one embodiment, one or more V, D and J gene segments, or one or more V and J gene segments are not rearranged. In one embodiment, one or more V, D and J gene segments, or one or more V and J gene segments are rearranged. In one embodiment, after rearrangement of one or more V, D and J gene segments, or one or more V and J gene segments, the mouse has a mouse ADAM6 gene or a homologue or ortholog or functional fragment thereof in its genome. It contains at least one encoding nucleic acid sequence. In one embodiment, after rearrangement, the mouse contains in its genome at least two nucleic acid sequences encoding the mouse ADAM6 gene or a homologue or ortholog or functional fragment thereof. In one embodiment, the mouse after rearrangement contains in its genome at least one nucleic acid sequence encoding a mouse ADAM6 gene or a homologue or ortholog or functional fragment thereof. In one embodiment, the mouse contains the ADAM6 gene of B cells or a homologue or ortholog or functional fragment thereof. In one embodiment, the mouse contains a non-B cell ADAM6 gene or a homologue or ortholog or functional fragment thereof.

In one aspect, there is provided a mouse expressing a human immunoglobulin heavy chain variable region or a functional fragment thereof from an endogenous mouse immunoglobulin heavy chain locus, wherein the mouse comprises functional ADAM6 activity in a male mouse.

In one embodiment, the male mouse comprises a single unmodified endogenous ADAM6 allele or an ortholog or homolog or functional fragment thereof at the endogenous ADAM6 site.

In one embodiment, the male mouse contains an ectopic mouse ADAM6 sequence encoding a protein conferring ADAM6 function, or a homologue or ortholog or functional fragment thereof.

In an embodiment, the male mice are located, for example, V gene segment sequence of the 3 'and the beginning of the D gene segment of the 5' ADAM6 sequence or a homologue or ohsol log on in the position and approximate mouse genome of the endogenous mouse ADAM6 allele Or a functional fragment.

In one embodiment, the male mouse is an ADAM6 sequence or a homologue or ortholog thereof flanked upstream, downstream, or upstream and downstream of the nucleic acid sequence encoding the immunoglobulin variable gene segment (with respect to the transcriptional direction of the ADAM6 sequence). Or a functional fragment. In a specific embodiment, the immunoglobulin variable gene segment is a human gene segment. In one embodiment, the immunoglobulin variable gene segment is a human gene segment, and the sequence encoding mouse ADAM6 or its ortholog or homolog or fragment functionality in the mouse is located between the human V gene segments; In one embodiment, the mouse comprises two or more human V gene segments, the sequence being located between the last V gene segment and the last to second V gene segment; In one embodiment, the sequence is at a position after the last V gene segment and the first D gene segment.

In one embodiment, the male mouse comprises an ADAM6 sequence or a homolog or ortholog or functional fragment thereof located at a position within the same or substantially the same endogenous immunoglobulin site as the wild-type male mouse. In specific embodiments, the endogenous site cannot encode a heavy chain variable region of an antibody, wherein the variable region comprises or is derived from an endogenous non-human gene segment. In a specific embodiment, the endogenous site is located at a position in the genome of a male mouse that disables the coding heavy chain variable region of the antibody. In various embodiments, the male mouse comprises an ADAM6 sequence located on the same chromosome as a human immunoglobulin gene segment, and the ADAM6 sequence encodes a functional ADAM6 protein.

In one aspect, there is provided a male mouse comprising a deletion of an endogenous ADAM6 gene or a non-functional endogenous ADAM6 gene in the germline; Here, the sperm cells of the mouse can move the fallopian tubes of the female mouse to fertilize the mouse's egg.

In one aspect, a male mouse is provided comprising a modification to a functional endogenous ADAM6 gene and an endogenous immunoglobulin heavy chain site. In one embodiment, the modification is made downstream or 3'of the endogenous ADAM6 gene. In one embodiment, the modification is the replacement of one or more endogenous immunoglobulin heavy chain gene segments with one or more human immunoglobulin heavy chain gene segments. In one embodiment, the modification is the insertion of one or more human immunoglobulin heavy chain gene segments upstream of the endogenous immunoglobulin heavy chain constant region gene.

In one aspect, a mouse is provided comprising a genetic modification that reduces endogenous mouse ADAM6 function, wherein the mouse is fully or partially functional by an endogenous unmodified allele (e.g., heterozygote), or in a male mouse. Contains at least some ADAM6 functionality by expression from an ectopic sequence encoding a typical ADAM6 or an ortholog or homolog or functional fragment thereof.

In one embodiment, the mouse contains sufficient ADAM6 function to confer to the male mouse the ability to produce offspring by mating compared to a male mouse lacking functional ADAM6. In one embodiment, ADAM6 function is conferred by the presence of an ectopic nucleotide sequence encoding mouse ADAM6 or a homologue or ortholog or functional fragment thereof. In one embodiment, the ADAM6 function is conferred by the endogenous ADAM6 gene present at the endogenous immunoglobulin site, wherein the endogenous immunoglobulin site refracts the coding heavy chain variable region of the antibody. Functional ADAM6 homologues or orthologs or fragments thereof in male mice partially or partially compensate for the loss of ability to produce progeny observed in male mice lacking sufficient endogenous mouse ADAM6 activity, e.g., the loss observed in ADAM6 knockout mice. It includes restoring to. In this sense, ADAM6 knockout mice contain endogenous sites or fragments thereof, but are not functional, i.e., do not express ADAM6 (ADAM6a and/or ADAM6b) at all or exhibit essentially normal ability to produce offspring of wild-type male mice. Include mice expressing ADAM6 (ADAM6a and/or ADAM6b) at insufficient levels to support. Loss of function, for example, in the structural gene of the locus (i.e., in the ADAM6a or ADAM6b coding region) or in the regulatory region of the locus (e.g., 5'for the ADAM6 gene or the 3'sequence of the ADAM6a or ADAM6b coding region) , Wherein the sequence controls the transcription of the ADAM6 gene, the expression of ADAM6 RNA, or the expression of the ADAM6 protein in whole or in part). In various embodiments, functional orthologs or homologs or fragments thereof in male mice are capable of fertilizing mouse oocytes by migrating the sperm of the male mouse (or most of the sperm cells in the ejaculated semen of the male mouse) by moving the mouse oviduct. These are things that make it possible.

In one embodiment, a male mouse expressing a human immunoglobulin variable region or a functional fragment thereof comprises sufficient ADAM6 activity to confer to the male mouse the ability to produce offspring by mating with a female mouse, and in one embodiment In that, male mice are at least 25% in one embodiment, at least 30% in one embodiment, at least 40% in one embodiment, at least 50% in one embodiment, and at least 60% in one embodiment. , Of a mouse carrying at least 70% in one embodiment, at least 80% in one embodiment, at least 90% in one embodiment, and in one embodiment one or two endogenous unmodified ADAM6 alleles. It exhibits the ability to produce offspring when mated with approximately the same female mouse.

In one embodiment, the male mouse expresses sufficient ADAM6 (or an ortholog or homolog or functional fragment thereof) so that sperm cells from the male mouse can cross the female mouse oviduct and fertilize the mouse egg.

In one embodiment, ADAM6 functionality is conferred by a nucleic acid sequence contiguous with the mouse chromosome sequence (e.g. , the nucleic acid is randomly integrated into the mouse chromosome; or, e.g., by targeting the nucleic acid to a specific location, e.g., site-specific. Enemy recombinase-mediated (e.g., Cre-mediated) is placed at a specific location by insertion or homologous recombination). In one embodiment, the ADAM6 sequence is on a nucleic acid separate from the chromosome of the mouse (e.g., the ADAM6 sequence is on an episome, i.e., extrachromosomally, e.g., an expression construct, vector, YAC, It is present in transchromosomes, etc.).

In one aspect, genetically modified mice and cells comprising modification of an endogenous immunoglobulin heavy chain locus are provided, wherein the mouse expresses at least a portion of an immunoglobulin heavy chain sequence, such as at least a portion of a human sequence, wherein the mouse is And functional ADAM6 activity in male mice. In one embodiment, the modification reduces or eradicates ADAM6 activity in the mouse. In one embodiment, the mouse is modified such that the two alleles encoding ADAM6 activity express or absent ADAM6, which is substantially unable to function to support normal mating in male mice. In one embodiment, the mouse further comprises an ectopic nucleic acid sequence encoding an encoding mouse ADAM6 or an ortholog or homolog or functional fragment thereof. In one embodiment, the modification retains ADAM6 activity in the mouse, making it impossible to encode the heavy chain variable region of the antibody at the endogenous immunoglobulin heavy chain locus. In a specific embodiment, the modification comprises a chromosomal inversion and or translocation that makes it impossible to arrange to encode a heavy chain variable region of an antibody operably linked to a heavy chain constant region in an endogenous immunoglobulin heavy chain variable gene segment.

In one aspect, genetically modified mice and cells comprising modification of an endogenous immunoglobulin heavy chain site are provided, wherein the modification reduces or eliminates ADAM6 activity expressed from the ADAM6 sequence of the site, and the mouse is an ADAM6 protein or Its orthologs or homologs or functional fragments are included. In various embodiments, the ADAM6 protein or fragment thereof is encoded by an ectopic ADAM6 sequence. In various embodiments, the ADAM6 protein or fragment thereof is expressed from an endogenous ADAM6 allele. In various embodiments, the mouse comprises a first immunoglobulin heavy chain allele comprising a first modification that reduces or eliminates expression of functional ADAM6 from the first immunoglobulin heavy chain allele, and the mouse comprises a second immunoglobulin heavy chain And a second immunoglobulin heavy chain allele comprising a second modification that does not substantially reduce or eliminate the expression of functional ADAM6 from the allele.

In various embodiments, the modification is the insertion of one or more human immunoglobulin heavy chain gene segments upstream or 5′ of the endogenous immunoglobulin heavy chain constant region gene. In various embodiments, the modification retains the endogenous ADAM6 gene located at the endogenous immunoglobulin heavy chain site.

In one embodiment, the second modification is located 3'of the last mouse V gene segment (with respect to the transcriptional direction of the mouse V gene segment) and is also a mouse (or chimeric human/mouse) immunoglobulin heavy chain constant gene or fragment thereof ( For example, a nucleic acid sequence encoding an encoding human and/or mouse: C _H 1 and/or hinge and/or C _H 2 and/or C _H 3 ).

In one embodiment, the modification is in the first immunoglobulin heavy chain allele at the first site encoding the first ADAM6 allele, and the ADAM6 function is the second immunoglobulin heavy chain allele at the second site encoding the functional ADAM6. Due to the expression of endogenous ADAM6 in the gene, wherein the second immunoglobulin heavy chain allele comprises at least one modification of the V, D and/or J gene segments. In a specific embodiment, at least one modification of the V, D and or J gene segment is a deletion, replacement with a human V, D, and/or J gene segment, to a camelid V, D, and/or J gene segment. Replacement of, humanized or camelized V, D, and/or J gene segments, replacement of heavy chain sequences with light chain sequences, and combinations thereof. In one embodiment, the at least one modification is a deletion of one or more heavy chain V, D, and/or J gene segments at the heavy chain locus and one or more light chain V and/or J gene segments (e.g., human light chain V and/or Or J gene segment).

In one embodiment, the modification is in the first immunoglobulin heavy chain allele at the first site and the second immunoglobulin heavy chain allele at the second site, and the ADAM6 function is ectopic at the non-immunoglobulin site in the germline of the mouse. It results from the expression of ADAM6. In a specific embodiment, the non-immunoglobulin site is the ROSA26 site. In a specific embodiment, the non-immunoglobulin site is transcriptionally active in regenerative tissue.

In one embodiment, the modification is in the first immunoglobulin heavy chain allele at the first site and the second immunoglobulin heavy chain allele at the second site, and the ADAM6 function is derived from the endogenous ADAM6 gene in the germline of the mouse. In a specific embodiment, the endogenous ADAM6 gene is juxtaposed with a mouse immunoglobulin gene segment.

In one embodiment, the modification is in the first immunoglobulin heavy chain allele at the first site and the second immunoglobulin heavy chain allele at the second site, and the ADAM6 function is ectopic ADAM6 in the first immunoglobulin heavy chain allele. It is due to the expression of the sequence. In one embodiment, the modification is in the first immunoglobulin heavy chain allele at the first site and the second immunoglobulin heavy chain allele at the second site, and the ADAM6 function or activity is in the second immunoglobulin heavy chain allele. It is due to the expression of ectopic ADAM6.

In one aspect, a mouse comprising a heterozygous or homozygous knockout of ADAM6 is provided. In one embodiment, the mouse further comprises a human or humanized immunoglobulin sequence, or a modified immunoglobulin sequence that is a camelid or camelized human or mouse immunoglobulin sequence. In one embodiment, the modified immunoglobulin sequence is at an endogenous heavy chain immunoglobulin site. In one embodiment, the modified immunoglobulin sequence comprises a human heavy chain variable gene sequence at an endogenous heavy chain immunoglobulin site. In one embodiment, the human heavy chain variable gene sequence replaces the endogenous heavy chain variable sequence at the endogenous immunoglobulin heavy chain site.

In one aspect, a mouse is provided that makes it impossible to express functional endogenous mouse ADAM6 from the endogenous mouse ADAM6 locus. In one embodiment, the mouse comprises an ectopic nucleic acid sequence encoding ADAM6, or a functional fragment thereof, which is functional in the mouse. In a specific embodiment, the ectopic nucleic acid sequence encodes a protein that rescues the loss of the ability to produce offspring exhibited by male mice that are homozygous for ADAM6 knockout. In a specific embodiment, the ectopic nucleic acid sequence encodes a mouse ADAM6 protein.

In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 site and comprises an ectopic nucleic acid sequence that confers mouse ADAM6 function. In one embodiment, the nucleic acid sequence comprises an endogenous mouse ADAM6 sequence or a functional fragment thereof. In one embodiment, the endogenous mouse ADAM6 sequence is located in a wild-type mouse between the 3′-most mouse immunoglobulin heavy chain V gene segment (V _H ) and the 5′-most mouse immunoglobulin heavy chain D gene segment (D _{H ).} ADAM6a- and ADAM6b-encoding sequences are included,

In one embodiment, the nucleic acid sequence comprises a sequence encoding mouse ADAM6a or a functional fragment thereof and/or a sequence encoding mouse ADAM6b or a functional fragment thereof, wherein ADAM6a and/or ADAM6b or functional fragment(s) thereof are Operably linked to a promoter. In one embodiment, the promoter is a human promoter. In one embodiment, the promoter is a mouse ADAM6 promoter. In a specific embodiment, the ADAM6 promoter is a sequence located between the first codon of the first ADAM6 gene closest to the _{mouse 5'-most D H} gene segment and the recombination signal sequence of the _{5'-most D H gene segment.} Including, wherein 5'is indicated with respect to the direction of transcription of the mouse immunoglobulin gene. In one embodiment, the promoter is a viral promoter. In a specific embodiment, the viral promoter is a cytomegalovirus (CMV) promoter. In one embodiment, the promoter is a ubiquitin promoter.

In one embodiment, the promoter is an inducible promoter. In one embodiment, the inducible promoter regulates expression in non-renewable tissue. In one embodiment, the inducible promoter regulates expression in regenerative tissue. In a specific embodiment, the expression of the mouse ADAM6a and/or ADAM6b sequence or functional fragment(s) thereof is progressively regulated by an inducible promoter in the regenerative tissue.

In one embodiment, the mouse ADAM6a and/or ADAM6b is selected from ADAM6a of SEQ ID NO: 1 and/or DAM6b of SEQ ID NO: 2. In one embodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO: 3. In a specific embodiment, the mouse ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO: 3 immediately upstream of the first codon of ADAM6a (with respect to the residue direction of ADAM6a, which extends upstream of the ADAM6 coding region to the end of SEQ ID NO: 3 In another specific embodiment, the ADAM6 promoter is a fragment extending from about 5 to less than about 20 nucleotides upstream of the start codon of ADAM6a to at least about 0.5 kb, 1 kb, 2 kb, or 3 kb upstream of the start codon of ADAM6a. .

In one embodiment, the nucleic acid sequence is SEQ ID NO: 3 or a fragment thereof that improves fertility or restores fertility to approximately wild-type fertility when placed in a mouse with low fertility due to infertility or lack of ADAM6. Includes. In one embodiment, SEQ ID NO: 3 or a fragment thereof confers to the male mouse the ability to produce sperm cells capable of traversing the female mouse oviduct to fertilize the mouse egg.

In one embodiment, the nucleic acid sequence is a sequence coding ADAM6 gene or a homolog or an ortholog or functional fragment thereof that produces a level of fertility that is the same as or comparable to that of a wild-type mouse when placed or maintained in a mouse. Sequence. Exemplary levels of fertility can be demonstrated by the ability of a male mouse to produce sperm cells capable of traversing the female mouse oviduct to fertilize a mouse egg.

In one aspect, deletion of the endogenous nucleotide sequence encoding the ADAM6 protein, replacement of the endogenous mouse V _H gene segment with a human V _H gene segment, and functional mouse ADAM6 protein or an ortholog or homolog or fragment thereof in a male mouse A mouse comprising an ectopic nucleotide sequence encoding is provided.

In one embodiment, the mouse comprises an immunoglobulin heavy chain locus comprising a deletion of an endogenous immunoglobulin locus nucleotide sequence comprising an endogenous ADAM6 gene, and comprises a nucleotide sequence encoding one or more human immunoglobulin gene segments, , Wherein the ectopic nucleotide sequence encoding the mouse ADAM6 protein is within or immediately adjacent to the nucleotide sequence encoding one or more human immunoglobulin gene segments.

In one embodiment, the mouse comprises replacement of all or substantially all of the _{endogenous V H gene segment with a nucleotide sequence encoding one or more human V H} gene segments, wherein the ectopic nucleotide sequence encoding the mouse ADAM6 protein is Within or immediately adjacent to the nucleotide sequence encoding one or more human V _{H gene segments.} In one embodiment, the mouse further comprises _{replacement of one or more endogenous D H} gene segments with one or more human D _H gene segments at the _{endogenous D H locus.} In one embodiment, the mouse further comprises _{replacement of one or more endogenous J H} gene segments with one or more human J _H gene segments at the _{endogenous J H locus.} In one embodiment, the mouse is the replacement of all or substantially all of the _{endogenous V H} , D _H and J _H gene segments and human V _H , D _H and J _{H at the endogenous V H} , D _H and J _{H locus.} Replacement with a gene segment, wherein the mouse comprises an ectopic sequence encoding a mouse ADAM6 protein. In one embodiment, the mouse comprises _{an insert of human V H} , D _H and J _H genes at the endogenous immunoglobulin heavy chain locus, wherein the mouse comprises a functional ADAM6 gene in the mouse. In a specific embodiment, a mouse ectopic sequence encoding the protein is present ADAM6 human V _H gene segment two of the human V _H gene segment present in the first 3 ' most of the V _H gene segment of the last 3 'end of the It is placed between the _{V H gene segments.} In a specific embodiment, the mouse contains an ectopic nucleotide sequence encoding a mouse ADAM6 protein comprising the deletion of all or substantially all of the _{mouse V H gene segment and replacement of all or substantially all of the human V H gene segment.} Is present downstream of the human gene segment V _H 1-2 and upstream of the human gene segment V _{H 6-1.}

In a specific embodiment, the mouse comprises replacement of all or substantially all of the _{endogenous V H gene segment with a nucleotide sequence encoding one or more human V H} gene segments, and an ectopic nucleotide sequence encoding a mouse ADAM6 protein. Is within or immediately adjacent to the nucleotide sequence encoding one or more human V _{H gene segments.}

In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is present on a transgene in the genome of the mouse. In one embodiment, the ectopic nucleotide sequence encoding the mouse ADAM6 protein is extrachromosomal in the mouse.

In one aspect, a mouse is provided comprising a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse expresses a B cell comprising a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence, and the B cell Contains a functional gene encoding ADAM6 or an ortholog or homolog or fragment thereof in a male mouse in its genome (eg, on a B cell chromosome). In one embodiment, a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence comprises a human heavy chain V, D, and/or J sequence; Mouse heavy chain V, D, and/or J sequences; Human or mouse light chain V and/or J sequences. In one embodiment, the heavy chain constant region gene sequence comprises a human or mouse heavy chain sequence selected from the group consisting _{of C H} 1, hinge, C _H 2, C _{H 3, and combinations thereof.}

In one aspect, a mouse is provided comprising a functionally silent endogenous immunoglobulin heavy chain variable locus, wherein ADAM6 function is maintained in the mouse, and at least one human immunity upstream or 5'of one or more mouse heavy chain constant regions is provided. It further comprises an insert of a globulin gene segment. In one embodiment, the at least one human immunoglobulin gene segment comprises at least one human V _H gene segment, at least one human D _H gene segment, and at least one human J _H gene segment. In a specific embodiment, the mouse further comprises an endogenous light chain locus that is functionally silent, wherein the mouse contains ADAM6 activity identical or comparable to that of the wild-type mouse, and also has one or more upstream or 5'of the mouse light chain constant region. It further comprises an insert of a human λ light chain gene segment. In one embodiment, the human λ light chain gene segment comprises 12 human Vλ gene segments and one or more human Jλ gene segments. In one embodiment, the human λ light chain gene segment comprises 12 human Vλ gene segments and 4 human Jλ gene segments. In one embodiment, the human λ light chain gene segment comprises 28 human Vλ gene segments and one or more human Jλ gene segments. In one embodiment, the human λ light chain gene segment comprises 28 human Vλ gene segments and 4 human Jλ gene segments. In one embodiment, the human λ light chain gene segment comprises 40 human Vλ gene segments and one or more human Jλ gene segments. In one embodiment, the human λ light chain gene segment comprises 40 human Vλ gene segments and 4 human Jλ gene segments. In various embodiments, the four human Jλ gene segments comprise Jλ1, Jλ2, Jλ3, and Jλ7. In various embodiments, the mouse light chain constant region is mouse Cκ or mouse Cλ.

In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a functionally silent immunoglobulin light chain gene and at least one human immunoglobulin heavy chain variable region gene of one or more endogenous immunoglobulin heavy chain variable region gene segments. And the replacement with a segment, wherein the mouse lacks a functional endogenous ADAM6 site, and the mouse contains an ectopic nucleotide sequence expressing a functional mouse ADAM6 protein or an ortholog or homolog or fragment thereof in a male mouse.

In one aspect, there is provided a mouse comprising an ectopic nucleotide sequence or a functional fragment of a mouse ADAM6 site or sequence that lacks a functional endogenous mouse ADAM6 site or sequence and encodes a mouse ADAM6 site, wherein the mouse has an ectopic ADAM6 site or sequence. It allows mating with mice of the opposite sex to produce a containing offspring. In one embodiment, the mouse is male. In one embodiment, the mouse is female.

In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a human immunoglobulin heavy chain variable region gene segment at an endogenous mouse immunoglobulin heavy chain variable region locus, and the mouse is at an endogenous mouse immunoglobulin heavy chain variable region locus. It lacks the endogenous functional ADAM6 sequence, and the mouse contains an ectopic nucleotide sequence that expresses a functional mouse ADAM6 protein or an ortholog or homolog or fragment thereof in a male mouse.

In one embodiment, the ectopic nucleotide sequence expressing the mouse ADAM6 protein is extrachromosomal. In one embodiment, the ectopic nucleotide sequence expressing the mouse ADAM6 protein is targeted at one or more sites in the genome of the mouse. In specific embodiments, the one or more sites comprise immunoglobulin sites.

In one aspect, a mouse expressing an immunoglobulin heavy chain sequence from a modified endogenous mouse immunoglobulin heavy chain locus is provided, wherein the heavy chain is derived from a human V gene segment, a D gene segment, and a J gene segment, wherein the mouse is a mouse It includes functional ADAM6 activity in.

In one embodiment, the mouse comprises a plurality of human V gene segments, a plurality of D gene segments, and a plurality of J gene segments. In one embodiment, the D gene segment is a human D gene segment. In one embodiment, the J gene segment is a human J gene segment. In one embodiment, the mouse further comprises a humanized heavy chain constant region sequence, wherein the humanization comprises replacement of a sequence selected from _{C H} 1, hinge, C _H 2, C _{H 3, and combinations thereof.} In a specific embodiment, the heavy chain is derived from a human V gene segment, a human D gene segment, a human J gene segment, a human C _H 1 sequence, a human or mouse hinge sequence, a mouse C _H 2 sequence, and a mouse C _H 3 sequence. . In another specific embodiment, the mouse further comprises a human light chain constant sequence.

In one embodiment, the mouse comprises an ADAM6 gene that is 5'and 3'flanked by an endogenous immunoglobulin heavy chain gene segment. In a specific embodiment, the endogenous immunoglobulin heavy chain gene segment disables the encoding of the heavy chain of the antibody. In a specific embodiment, the ADAM6 gene of the mouse is in the same position as that in the wild-type mouse, and the endogenous immunoglobulin heavy chain variable locus of the mouse makes it impossible to rearrange to encode the heavy chain of the antibody.

In one embodiment, the V gene segment is 5'flanked by a sequence encoding functional ADAM6 activity in a mouse (with respect to transcription of the V gene segment).

In one embodiment, the V gene segment is 3'flanked by a sequence encoding functional ADAM6 activity in a mouse (with respect to transcription of the V gene segment).

In one embodiment, the D gene segment is flanked by a sequence encoding functional ADAM6 activity in a mouse (with respect to transcription of the D gene segment).

In one embodiment, functional ADAM6 activity in mice is 5'of the modified endogenous mouse heavy chain immunoglobulin locus-5'and 3'of most D gene segments-3'of most V gene segments ( With respect to the direction of transcription).

In one embodiment, functional ADAM6 activity in mice is due to the expression of a nucleotide sequence located between two human V gene segments at the modified endogenous mouse heavy chain immunoglobulin site. In one embodiment, the two human V gene segments are a human V _H 1-2 gene segment and a V _H 6-1 gene segment.

In one embodiment, the nucleotide sequence comprises a sequence selected from a mouse ADAM6b sequence or a functional fragment thereof, a mouse ADAM6a sequence or a functional fragment thereof, and combinations thereof.

In one embodiment, the nucleotide sequence between two human V gene segments is positioned in opposite transcriptional orientation with respect to the human V gene segment. In a specific embodiment, the nucleotide sequence is encoded from 5'to 3'with respect to the direction of transcription of the ADAM6 gene, followed by the ADAM6a sequence, followed by the ADAM6b sequence.

In one embodiment, the mouse comprises _{replacement of a human ADAM6 pseudogene sequence between V H} 1-2 and V _H 6-1, which is a human V gene segment, with a mouse ADAM6 sequence or a functional fragment thereof.

In one embodiment, the sequence encoding functional ADAM6 activity in a mouse is a mouse ADAM6 sequence or a functional fragment thereof.

In one embodiment, the mouse comprises an endogenous mouse DFL16.1 gene segment (eg, in a mouse that is heterozygous for a modified endogenous mouse immunoglobulin heavy chain locus), or a human D _H 1-1 gene segment. In one embodiment, the D gene segment of the immunoglobulin heavy chain expressed by the mouse is derived from an _{endogenous mouse DFL16.1 gene segment or a human D H 1-1 gene segment.}

In one aspect, it comprises a nucleic acid sequence encoding mouse ADAM6 (or a homologue or ortholog or functional fragment thereof) in a DNA-bearing cell of a non-arranged B cell lineage, but A mouse is provided that does not contain a nucleic acid sequence encoding mouse ADAM6 (or a homologue or ortholog or functional fragment thereof) in a B cell containing an aligned immunoglobulin site, wherein mouse ADAM6 (or a homologue or ortholog thereof Alternatively, a nucleic acid sequence encoding a functional fragment) is found in the genome at a location different from the location where the mouse ADAM6 gene appears in wild-type mice. In one embodiment, the nucleic acid sequence encoding mouse ADAM6 (or homolog or ortholog or functional fragment thereof) is present in all or substantially all of the DNA-bearing cells that are not rearranged B cell lineages; In one embodiment, the nucleic acid sequence is present in the germline cells of the mouse rather than the chromosome of the rearranged B cells.

In one aspect, a nucleic acid sequence encoding mouse ADAM6 (or a homologue or an ortholog or functional fragment thereof) is provided in all or substantially all of the DNA-bearing cells, including B cells containing the rearranged immunoglobulin site. A mouse comprising is provided wherein the nucleic acid sequence encoding mouse ADAM6 (or a homologue or ortholog or functional fragment thereof) occurs in the genome at a location different from the location in which the mouse ADAM6 gene appears in wild-type mice. In one embodiment, the nucleic acid sequence encoding mouse ADAM6 (or homolog or ortholog or functional fragment thereof) is on a nucleic acid contiguous with the rearranged immunoglobulin site. In one embodiment, the nucleic acid contiguous with the rearranged immunoglobulin site is a chromosome. In one embodiment, the chromosome is a chromosome found in wild-type mice, which chromosome comprises a modification of a mouse immunoglobulin site.

In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a B cell comprising an ADAM6 sequence or an ortholog or homolog thereof in its genome. In one embodiment, the ADAM6 sequence or an ortholog or homolog thereof is at the site of an immunoglobulin heavy chain. In one embodiment, the ADAM6 sequence or an ortholog or homolog thereof is at a site other than the immunoglobulin site. In one embodiment, the ADAM6 sequence is on a transgene driven by a heterologous promoter. In a specific embodiment, the non-homologous promoter is a non-immunoglobulin promoter. In a specific embodiment, the B cell expresses the ADAM6 protein or an ortholog or homolog thereof.

In one embodiment, 90% or more of the mouse B cells contain a functional gene-encoding ADAM6 protein or an ortholog thereof or a homologue thereof or a fragment thereof in the mouse. In a specific embodiment, the mouse is a male mouse.

In one embodiment, the B cell genome comprises a first allele and a second allele comprising the ADAM6 sequence or an ortholog or homolog thereof. In one embodiment, the B cell genome comprises a first allele but not a second allele comprising the ADAM6 sequence or an ortholog or homolog thereof.

In one aspect, the mouse is provided with a mouse comprising a modification in at least one endogenous immunoglobulin heavy chain allele, wherein the modification retains at least one endogenous ADAM6 allele, and the mouse is upstream of the mouse light chain constant region. And an insert of at least one human Vλ gene segment and at least one human Jλ gene segment. In various embodiments, the mouse light chain constant region is mouse Cκ or mouse Cλ.

In one embodiment, the modification makes it impossible for the mouse to express a functional heavy chain comprising an endogenous heavy chain gene segment rearranged from at least one heavy chain allele, and is also located within at least one endogenous immunoglobulin heavy chain allele. It retains the endogenous ADAM6 allele.

In one embodiment, the mouse is incapable of expressing a functional heavy chain comprising an endogenous heavy chain gene segment rearranged from at least one of the endogenous immunoglobulin heavy chain alleles, and the mouse is unable to express the ADAM6 protein from the endogenous ADAM6 allele. Manifest. In a specific embodiment, the mouse is unable to express a functional heavy chain comprising an endogenous heavy chain gene segment rearranged from two endogenous immunoglobulin heavy chain alleles, and the mouse is an ADAM6 protein from one or more endogenous ADAM6 alleles. Express.

In one embodiment, the mouse makes it impossible to express the functional heavy chain from each endogenous heavy chain allele, and the mouse is 1, 2, (with respect to the transcription direction of the mouse heavy chain site) of the mouse immunoglobulin heavy chain constant region sequence. 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 or more Mbps upstream of a functional ADAM6 allele. In a specific embodiment, the functional ADAM6 allele is at the endogenous immunoglobulin heavy chain site (e.g., in an intergenic VD region, between two V gene segments, between a V gene segment and a D gene segment, a D gene segment and a J gene segment). In between, etc.). In a specific embodiment, the functional ADAM6 allele is located within a 90-100 kb intergenic sequence between the last mouse V gene segment and the first mouse D gene segment.

In one aspect, a mouse is provided comprising a modification in one or more endogenous ADAM6 alleles.

In one embodiment, the modification renders it impossible for the mouse to express a functional ADAM6 protein from at least one of the one or more endogenous ADAM6 alleles. In a specific embodiment, the mouse is unable to express the functional ADAM6 protein from each of the endogenous ADAM6 alleles.

In one embodiment, the mouse is incapable of expressing the functional ADAM6 protein from each endogenous ADAM6 allele.

In one embodiment, the mouse is unable to express the functional ADAM6 protein from each endogenous ADAM6 allele, and the mouse is 1, 2, of the mouse immunoglobulin heavy chain constant region sequence (with respect to the transcriptional direction of the mouse heavy chain locus). 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 or more kb upstream. In a specific embodiment, the ectopic ADAM6 sequence is at an endogenous heavy chain locus (e.g., in an intergenic VD region, between two V gene segments, between a V gene segment and a D gene segment, between a D gene segment and a J gene segment, etc.) ) have. In a specific embodiment, the ectopic ADAM6 sequence is located within a 90-100 kb intergenic sequence between the last mouse V gene segment and the first D gene segment. In another specific embodiment, the endogenous 90-100 kb intergenic V-D sequence is removed, and the ectopic ADAM6 sequence is positioned between the last gene segment and the first D gene segment.

In one aspect, a sterile male mouse is provided, wherein the mouse comprises deletion of two or more endogenous ADAM6 alleles. In one aspect, a female mouse is provided that is a carrier of male infertility characteristics, wherein the female mouse comprises a knockout of a non-functional ADAM6 allele or an endogenous ADAM6 allele in its germline.

In one aspect, a mouse is provided comprising an endogenous immunoglobulin heavy chain V, D, and or J gene segment that makes it impossible to rearrange to encode a heavy chain of an antibody, wherein the majority of the mouse's B cells are functional ADAM6 Contains genes. In various embodiments, the majority of mouse B cells further comprise at least one human Vλ gene segment and at least one human Jλ gene segment upstream of the mouse immunoglobulin light chain constant region. In one embodiment, the mouse immunoglobulin light chain constant region is selected from mouse Cκ and mouse Cλ.

In one embodiment, the mouse comprises an intact endogenous immunoglobulin heavy chain V, D and J gene segments that make it impossible to rearrange to encode a functional heavy chain of the antibody. In one embodiment, the mouse comprises at least 1 to 89 V gene segments, at least 1 to 13 D gene segments, at least 1 to 4 J gene segments, and combinations thereof; At least 1 to 89 V gene segments, at least 1 to 13 D gene segments, and at least 1 to 4 J gene segments are impossible to rearrange to encode the heavy chain variable region of the antibody. Let's do it. In a specific embodiment, the mouse comprises a functional ADAM6 gene located within the intact endogenous immunoglobulin heavy chain V, D and J gene segments. In one embodiment, the mouse comprises an endogenous heavy chain locus comprising an endogenous ADAM6 locus, wherein the endogenous heavy chain locus comprises 89 V gene segments, 13 D gene segments and 4 J gene segments, and an endogenous heavy chain gene The segment makes it impossible to rearrange to encode the heavy chain variable region of the antibody, and the ADAM6 site encodes a functional ADAM6 protein in mice.

In one aspect, a mouse is provided that lacks endogenous immunoglobulin heavy chain V, D and J gene segments, wherein the majority of the mouse's B cells comprise the ADAM6 sequence or an ortholog or homolog thereof. In one embodiment, the majority of mouse B cells express an immunoglobulin light chain comprising a human lambda variable domain and an endogenous immunoglobulin light chain constant region.

In one embodiment, the mouse lacks an endogenous immunoglobulin heavy chain gene segment selected from two or more V gene segments, two or more D gene segments, two or more J gene segments, and combinations thereof. In one embodiment, the mouse is an immunoglobulin selected from at least 1 to 89 V gene segments, at least 1 to 13 D gene segments, at least 1 to 4 J gene segments, and combinations thereof. It lacks a heavy chain gene segment. In one embodiment, the mouse lacks a genomic DNA fragment from chromosome 12 comprising about 3 megabases of endogenous immunoglobulin heavy chain sites. In a specific embodiment, the mouse lacks all functional endogenous heavy chain V, D and J gene segments. In a specific embodiment, the mouse _{lacks 9 V H} gene segments, 13 D _H gene segments and 4 J _H gene segments.

In one aspect, a mouse is provided, wherein the mouse has a genome in the germline comprising a modification of an immunoglobulin heavy chain site, wherein the modification to the immunoglobulin heavy chain site is one or more non- Mouse immunoglobulin variable region sequence, wherein the mouse contains a nucleic acid sequence encoding a mouse ADAM6 protein. In a preferred embodiment, the D _H and J _H sequences of the immunoglobulin heavy chain locus and at least 3, at least 10, at least 20, at least 40, at least 60 or at least 80 V _H sequences are by non-mouse immunoglobulin variable region sequences. Is replaced. In a further preferred embodiment, the D _H , J _H and all V _H sequences of the immunoglobulin heavy chain site are replaced by a non-mouse immunoglobulin variable region sequence. The non-mouse immunoglobulin variable region sequence may be non-rearrangement. In a preferred embodiment, the non-mouse immunoglobulin variable region sequence comprises complete non-rearranged D _H and J _H regions of non-mouse species and at least 3, at least 10, at least 20, at least 40, at least 60 or at least 80 It includes a non-rearrangement V _H sequence. In a further preferred embodiment, the non-mouse immunoglobulin variable region sequence comprises a fully variable region comprising _{all V H} , D _H and J _{H regions of the non-mouse species.} The non-mouse species may be Homo sapiens, and the non-mouse immunoglobulin variable region sequence may be a human sequence.

In one aspect, a mouse expressing an antibody comprising at least one human variable domain/non-human constant domain immunoglobulin polypeptide is provided, wherein the mouse is a mouse ADAM6 protein or an ortholog thereof from a site other than the immunoglobulin site. Or it expresses a homologue.

In one embodiment, the ADAM6 protein or an ortholog or homolog thereof is expressed in a mouse B cell, wherein the B cell comprises a rearranged immunoglobulin sequence comprising a human variable sequence and a non-human constant sequence.

In one embodiment, the non-human constant sequence is a rodent sequence. In one embodiment, the rodent is selected from mice, rats and hamsters.

In one aspect, a method of constructing a sterile male mouse is provided, wherein the method provides non-functionality to the endogenous ADAM6 allele of the donor ES cell (or knocking out the allele), and the donor ES cell is host Introducing an embryo, fertilizing a host embryo into a surrogate mother, and causing the surrogate mother to produce a descendant wholly or partially derived from a donor ES cell. In one embodiment, the method further comprises crossing offspring to obtain infertile male mice.

In one aspect, a method of making a mouse having a genetic modification of interest is provided, wherein the mouse is infertile, the method comprising the steps of: (a) making a genetic modification of interest in the genome; (b) modifying the genome with knockout of the endogenous ADAM6 allele, or imparting non-functionality to the endogenous ADAM6 allele; And (c) using the genome when making the mouse. In various embodiments, the genome is derived from ES cells or used in nuclear transplant experiments.

In one aspect, a mouse constructed using a targeting vector, nucleotide construct or cell as described herein is provided.

In one aspect, a wild-type mouse of a mouse as described herein or a descendant by mating with a second genetically modified mouse is provided.

In one aspect, a method of maintaining a mouse strain is provided, wherein the mouse strain comprises replacement of a mouse immunoglobulin heavy chain sequence with one or more non-homologous immunoglobulin heavy chain sequences. In one embodiment, the at least one non-homologous immunoglobulin heavy chain sequence is a human immunoglobulin heavy chain sequence.

In one embodiment, the mouse strain comprises _{deletion of one or more mouse V H} , D _H , and/or J _H gene segments. In one embodiment, the mouse further comprises one or more human V _H gene segments, one or more human D _H gene segments, and/or one or more human J _H gene segments. In one embodiment, the mouse has at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 human V _H segments, at least 27 human D _H gene segments, and at least 6 J _H gene segments. Include

. In specific embodiments, the mouse comprises at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 human V _H segments, at least 27 human D _H gene segments, and at least 6 J _H gene segments. Is operably linked to a constant region gene. In one embodiment, the constant region gene is a mouse constant region gene. In one embodiment, the constant region gene comprises a mouse constant region gene sequence selected from _{C H} 1, hinge, C _H 2, C _H 3, and/or C _{H 4 or combinations thereof.}

In one embodiment, the method comprises the steps of generating a male mouse that is a heterozygous for replacement of the mouse immunoglobulin heavy chain sequence, and a wild-type female mouse or a female that is a heterozygous or a heterozygous for the human heavy chain sequence. And mating with a mouse. In one embodiment, the method comprises maintaining the variant by repeatedly crossing the heterozygous male with a homozygous or heterozygous or wild-type female for a human heavy chain sequence.

In one embodiment, the method comprises obtaining cells from a male or female mouse that is a homozygous or heterozygous for a human heavy chain sequence, and using these cells as a donor cell or a nucleus therefrom as a donor nucleus, and the cell Alternatively, it includes the step of making a genetically modified animal using the host cell using the nucleus and/or modifying the cell and/or the nucleus in the surrogate mother.

In one embodiment, only male mice that are heterozygous for replacement at the heavy chain site are crossed with female mice. In a specific embodiment, the female mouse is homozygous, heterozygous, or wild type with respect to the replaced heavy chain site.

In one embodiment, the mouse further comprises a replacement of a λ and/or κ light chain variable sequence with a non-homologous immunoglobulin light chain sequence at the endogenous immunoglobulin light chain locus. In one embodiment, the non-homologous immunoglobulin light chain sequence is a human immunoglobulin λ and/or κ light chain variable sequence.

In one embodiment, the mouse further comprises a transgene at a site other than the endogenous immunoglobulin site, wherein the transgene is operably linked to the immunoglobulin light chain constant region sequence (relative to the unrearranged) or For alignment) a sequence encoding a fused rearranged or unrearranged non-homologous λ or κ light chain sequence (e.g., unrearranged V _L and unrearranged J _L , or rearranged VJ) Includes. In one embodiment, the non-homologous λ or κ light chain sequence is human. In one embodiment, the constant region sequence is selected from rodents, humans and non-human primates. In one embodiment, the constant region sequence is selected from mouse, rat and hamster. In one embodiment, the transgene comprises a non-immunoglobulin promoter that drives the expression of the light chain sequence. In a specific embodiment, the promoter is a transcriptionally active promoter. In a specific embodiment, the promoter is the ROSA26 promoter.

In one aspect, a nucleic acid construct comprising an upstream homology section and a downstream homology section is provided, wherein the upstream homology section comprises a sequence identical or substantially identical to a human immunoglobulin heavy chain variable region sequence, and The stream homology section comprises a sequence that is identical or substantially identical to the human or mouse immunoglobulin variable region sequence and is positioned between the downstream homology sections and comprises a nucleotide sequence encoding a mouse ADAM6 protein. In a specific embodiment, the sequence encoding the mouse ADAM6 gene is operably linked to a mouse promoter, and thus the mouse ADAM6 is linked to a wild-type mouse.

In one aspect, (a) a nucleotide sequence identical to or substantially identical to a human variable region gene segment nucleotide sequence; And (b) a targeting vector comprising a nucleotide sequence encoding a functional mouse ADAM6 or an ortholog or homolog or fragment thereof in a mouse is provided.

In one embodiment, the targeting vector further comprises a promoter operably linked to a sequence encoding mouse ADAM6. In a specific embodiment, the promoter is a mouse ADAM6 promoter.

In one aspect, a nucleotide construct for modifying a mouse immunoglobulin heavy chain variable site is provided, wherein the construct comprises at least one site-specific recombinase recognition site and a functional ADAM6 protein or an ortholog or homolog thereof in the mouse. Or a sequence encoding a fragment.

In one aspect, mouse cells and mouse embryos are provided, including, but not limited to, ES cells, pluripotent cells and induced pluripotent cells, comprising genetic modifications as described herein. Cells that are XX and cells that are XY are also provided. The cells by transformation, e.g., pronuclear injection (pronuclear injection) as described herein comprising a core containing a modified introducing into the cells is also provided. Cells, embryos and mice comprising the ADAM6 gene introduced by the virus, such as cells, embryos and mice comprising a transduction construct comprising the ADAM6 gene functional in a mouse, are also provided.

In one aspect, a genetically modified mouse cell is provided, wherein the cell lacks a functional endogenous mouse ADAM6 site and the cell comprises an ectopic nucleotide sequence encoding a mouse ADAM6 protein or functional fragment thereof. In one embodiment, the cell further comprises a modification of the endogenous immunoglobulin heavy chain variable gene sequence. In a specific embodiment, the modification of the endogenous immunoglobulin heavy chain variable gene sequence comprises _{a deletion selected from a deletion of a mouse V H} gene segment, a deletion of a mouse D _H gene segment, a deletion of a mouse J _H gene segment, and combinations thereof. . In a specific embodiment, the mouse comprises _{replacement of one or more mouse immunoglobulin V H} , D _H , and/or J _H sequences with human immunoglobulin sequences. In a specific embodiment, the human immunoglobulin sequence is selected from human V _H , human Vλ, human D _H , human J _H , human J _L , and combinations thereof.

In one embodiment, the cell is a pluripotent cell, a pluripotent cell, or an induced pluripotent cell. In a specific embodiment, the cell is a mouse ES cell.

In one aspect, a mouse B cell is provided, wherein the mouse B cell comprises a rearranged immunoglobulin heavy chain gene, and the B cell encodes a functional ADAM6 protein or an ortholog or homolog or fragment thereof in a male mouse. The nucleic acid sequence is included on the chromosome of the B cell. In one embodiment, it comprises two alleles of a mouse B cell nucleic acid sequence.

In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (eg, B cell chromosome) contiguous with the rearranged mouse immunoglobulin heavy chain site.

In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (eg, B cell chromosome) separate from the nucleic acid molecule comprising the rearranged mouse immunoglobulin heavy chain site.

In one embodiment, the mouse B cell comprises a rearranged non-mouse immunoglobulin variable gene sequence operably linked to a mouse or human immunoglobulin constant region gene, wherein the B cell is a functional ADAM6 protein or It contains a nucleic acid sequence that encodes an ortholog or homolog or fragment thereof.

In one aspect, there is provided a body mouse cell comprising a chromosome comprising a modified immunoglobulin heavy chain site, and a nucleic acid sequence encoding a functional mouse ADAM6 or an ortholog or homolog or fragment thereof in a male mouse. In one embodiment, the nucleic acid sequence is on the same chromosome as the modified immunoglobulin heavy chain site. In one embodiment, the nucleic acid is on a different chromosome than the modified immunoglobulin heavy chain site. In one embodiment, the body cell comprises a single complex of nucleic acid sequences. In one embodiment, the body cell comprises at least two copies of the nucleic acid sequence. In a specific embodiment, the body cell is a B cell. In a specific embodiment, the cell is a germ cell. In a specific embodiment, the cell is a stem cell.

In one aspect, there is provided a mouse germ cell comprising a nucleic acid sequence encoding mouse ADAM6 (or a homologue or ortholog or functional fragment thereof) on a chromosome of the germ cell, but mouse ADAM6 (or a homologue or ortholog thereof) Alternatively, a nucleic acid sequence encoding a functional fragment) is located in a chromosome different from that of a wild-type mouse germ cell. In one embodiment, the nucleic acid sequence is at a mouse immunoglobulin site. In one embodiment, the nucleic acid sequence is on the same chromosome of a germ cell as the mouse immunoglobulin site. In one embodiment, the nucleic acid sequence is on a chromosome of a germ cell that is different from the mouse immunoglobulin site. In one embodiment, the mouse immunoglobulin site comprises replacement of at least one mouse immunoglobulin sequence with at least one non-mouse immunoglobulin sequence. In a specific embodiment, the at least one non-mouse immunoglobulin sequence is a human immunoglobulin sequence.

In one aspect, pluripotent, induced pluripotent or pluripotent cells derived from a mouse as described herein are provided. In a specific embodiment, the cell is a mouse embryonic stem (ES) cell.

In one aspect, a cell or tissue derived from a mouse as described herein is provided. In one embodiment, the cell or tissue is derived from the spleen, lymph node or bone marrow of a mouse as described herein. In one embodiment, the cell is a B cell. In one embodiment, the cell is an embryonic stem cell. In one embodiment, the cell is a germ cell.

In one embodiment, the tissue is selected from connective tissue, muscle tissue, nerve tissue, and epithelial tissue. In a specific embodiment, the tissue is a regenerated tissue.

In one embodiment, cells and/or tissues derived from mice as described herein are isolated for use in one or more in vitro assays. In various embodiments, the one or more in vitro assays include physical, thermal, electrical, mechanical or optical properties, surgical procedures, measurement of interactions of different tissue types, development of imaging techniques, or combinations thereof.

In an aspect, there is provided the use of a cell or tissue derived from a mouse as described herein for making an antibody. In one aspect, there is provided the use of cells or tissues derived from mice as described herein for making hybridomas or quadromas.

In one aspect, the non-human cell comprises a chromosome or fragment thereof of a non-human animal as described herein. In one embodiment, the non-human cell comprises the nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises a chromosome or fragment thereof as a result of nuclear transplantation.

In one aspect, a nucleus derived from a mouse as described herein is provided. In one embodiment, the nucleus is derived from a diploid cell rather than a B cell.

In one aspect, a nucleotide sequence encoding an immunoglobulin variable region constructed in a mouse as described herein is provided.

In one aspect, an immunoglobulin heavy chain or immunoglobulin light chain variable region amino acid sequence of an antibody produced in a mouse as described herein is provided.

In one aspect, an immunoglobulin heavy chain or immunoglobulin light chain variable region nucleotide sequence encoding the variable region of an antibody produced in a mouse as described herein is provided.

In one aspect, an antibody or antigen-binding fragment thereof produced in a mouse as described herein (eg, Fab, F(ab) ₂ , scFv) is provided.

In one aspect, a method of constructing a genetically modified mouse is provided, wherein the method comprises one or more immunoglobulin heavy chain gene segments (with respect to transcription of immunoglobulin heavy chain gene segments) upstream of the endogenous ADAM6 site of the mouse. Replacing the above human immunoglobulin heavy chain gene segments with one or more human immunoglobulin heavy chain or light chain gene segments (with respect to transcription of the immunoglobulin heavy chain gene segment) downstream of the ADAM6 site of the mouse. And replacing it with. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments upstream of the endogenous ADAM6 site of the mouse comprise a V gene segment. In one embodiment, the human immunoglobulin gene segment that replaces one or more endogenous immunoglobulin gene segments upstream upstream of the endogenous ADAM6 site of the mouse comprises V and D gene segments. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments downstream of the endogenous ADAM6 site of the mouse comprise a J gene segment. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments downstream of the endogenous ADAM6 site of the mouse comprise D and J gene segments. In one embodiment, the one or more human immunoglobulin gene segments that replace one or more endogenous immunoglobulin gene segments downstream of the endogenous ADAM6 site of the mouse comprise V, D and J gene segments.

In one embodiment, the one or more immunoglobulin heavy chain gene segments upstream and/or downstream of the ADAM6 gene are replaced with pluripotent, induced pluripotent, or pluripotent cells to form genetically modified progenitor cells; Genetically modified progenitor cells are introduced into the host; A host comprising a genetically modified progenitor cell is conceived to form a mouse comprising a genome derived from the genetically modified progenitor cell. In one embodiment, the host is an embryo. In a specific embodiment, the host is selected from a mouse pre-morula (eg , 8- or 4-cell phase), a tetraploid embryo, an aggregating agent of embryonic cells, or a blastocyst.

In one aspect, a method of constructing a genetically modified mouse is provided, wherein the method comprises a mouse nucleotide sequence comprising a mouse immunoglobulin gene segment and mouse ADAM6 (or its ortholog or homolog or fragment functionality in a male mouse). The step of replacing the nucleotide sequence with a sequence containing a human immunoglobulin gene segment to form a first chimeric site, followed by a mouse ADAM6-encoding sequence (or a sequence encoding an ortholog or homolog or a functional fragment thereof) Inserting into a sequence comprising a human immunoglobulin gene segment to form a second chimeric site.

In one embodiment, the second chimeric site comprises a human immunoglobulin heavy chain variable (V _H ) gene segment. In one embodiment, the second chimeric site comprises a human immunoglobulin light chain variable (V _L ) gene segment. In a specific embodiment, the second chimeric site comprises a human D _H gene segment and _{a human V H} gene segment or a human Vλ gene segment operably linked to a human _{J H gene segment.} In another specific embodiment, the second chimeric site operates on a human C _H 1 sequence, _{fused with a mouse C H} 2 + C _H 3 sequence, or a third chimeric site comprising _{human C H 1 and human hinge sequences} Connected possible.

In one aspect, there is provided a use of a mouse comprising an ectopic nucleotide sequence comprising a mouse ADAM6 locus or sequence for making a male fertile mouse, wherein the use comprises an ectopic nucleotide sequence comprising a mouse ADAM6 locus or sequence. The steps of mating a mouse with a functional endogenous mouse ADAM6 site or a mouse lacking the sequence, and obtaining a female or male progeny that can produce an ectopic ADAM6 site or a progeny having the sequence, and obtaining a male progeny containing the ectopic ADAM6 site or sequence. And, the male exhibits approximately the same fertility as that of a wild-type male mouse.

In one aspect, the use of a mouse as described herein for constructing an immunoglobulin variable region nucleotide sequence is provided.

In one aspect, there is provided the use of a mouse as described herein to construct a fully human Fab or fully human F(ab) _2.

In one aspect, there is provided the use of a mouse as described herein to construct an immortalized cell line.

In one aspect, the use of a mouse as described herein for making a hybridoma or quadroma is provided.

In one aspect, the use of a mouse as described herein for constructing a phage library containing a human heavy chain variable region and a human light chain variable region is provided.

In one aspect, there is provided a use of a mouse as described herein for generating a variable region sequence for making a human antibody, the use of which is (a) immunizing a mouse as described in the specification with an antigen of interest. (B) isolating lymphocytes from the immunized mice of (a), (c) exposing the lymphocytes to one or more labeled antibodies, (d) identifying lymphocytes capable of binding to the antigen of interest. And (e) generating the variable region sequence by amplifying the one or more variable region nucleic acid sequences from the FGU.

In one embodiment, the lymphocyte is derived from the spleen of a mouse. In one embodiment, the lymphocyte is derived from a lymph node in a mouse. In one embodiment, the lymphocyte is derived from the bone marrow of a mouse.

In one embodiment, the labeled antibody is a fluorophore-conjugated antibody. In one embodiment, the one or more fluorophore-conjugated antibodies are selected from IgM, IgG, and/or combinations thereof.

In one embodiment, the lymphocyte is a B cell.

In one embodiment, the one or more variable region nucleic acid sequences comprise a heavy chain variable region sequence. In one embodiment, the one or more variable region nucleic acid sequences comprise a light chain variable region sequence. In a specific embodiment, the light chain variable region sequence is an immunoglobulin κ light chain variable region sequence. In one embodiment, the at least one variable region nucleic acid sequence comprises a heavy chain and a κ light chain variable region sequence.

In one embodiment, there is provided the use of a mouse as described herein for generating heavy chain and κ light chain variable region sequences to produce a human antibody, the use of which comprises (a) a mouse as described herein. Immunizing with the antigen of interest, (b) isolating the spleen from the immunized mouse of (a), (c) exposing B lymphocytes from the spleen to one or more labeled antibodies, (d) the antigen of interest (C) identifying the B lymphocytes capable of binding to, and (e) generating heavy and κ light chain variable region sequences by amplifying a heavy chain variable region nucleic acid sequence and a κ light chain variable region nucleic acid sequence from B lymphocytes. Includes.

In one embodiment, there is provided the use of a mouse as described herein for generating heavy chain and κ light chain variable region sequences to produce a human antibody, the use of which comprises (a) a mouse as described herein. Immunizing with an antigen of interest, (b) isolating one or more lymph nodes from the immunized mouse of (a), (c) exposing B lymphocytes from one or more lymph nodes to one or more labeled antibodies, (d ) Identifying the B lymphocytes of (c) capable of binding to the antigen of interest, and (e) amplifying the heavy chain variable region nucleic acid sequence and the κ light chain variable region nucleic acid sequence from the B lymphocytes to obtain heavy and κ light chain variable region sequences. Including the step of generating.

In one embodiment, there is provided the use of a mouse as described herein for generating heavy chain and κ light chain variable region sequences to produce a human antibody, the use of which comprises (a) a mouse as described herein. Immunizing with an antigen of interest, (b) isolating the bone marrow from the immunized mouse of (a), (c) exposing B lymphocytes from the bone marrow to one or more labeled antibodies, (d) an antigen of interest (C) identifying the B lymphocytes capable of binding to, and (e) generating heavy and κ light chain variable region sequences by amplifying a heavy chain variable region nucleic acid sequence and a κ light chain variable region nucleic acid sequence from B lymphocytes. Includes. In various embodiments, the one or more labeled antibodies are selected from IgM, IgG, and/or combinations thereof.

In various embodiments, the use of a mouse as described herein for generating heavy and κ light chain variable region sequences to produce human antibodies is provided, wherein the use of amplified heavy and light chain variable region sequences is And fusing the light chain constant region sequence, exerting the fused heavy and light chain sequences in the cell, and recovering the expressed heavy and light chain sequences to generate a human antibody.

In various embodiments, the human heavy chain constant region is selected from IgM, IgD, IgA, IgE and IgG. In various specific embodiments, the IgG is selected from IgG1, IgG2, IgG3 and IgG4. In various embodiments, the human heavy chain constant region comprises C _H 1, hinge, C _H 2, C _H 3, C _H 4, or combinations thereof. In various embodiments, the light chain constant region is an immunoglobulin κ constant region. In various embodiments, the cells are HeLa cells, DU145 cells, Lncap cells, MCF-7 cells, MDA-MB-438 cells, PC3 cells, T47D cells, THP-1 cells, U87 cells, SHSY5Y (human neuroblastoma) cells. , Saos-2 cells, Vero cells, CHO cells, GH3 cells, PC12 cells, human retinal cells (eg, PER.C6™ cells), and MC3T3 cells. In a specific embodiment, the cell is a CHO cell.

In one aspect, provided is a method of generating a reverse-chimeric rodent-human antibody specific for an antigen of interest, the method comprising immunizing a mouse as described herein with an antigen, a reverse specific for the antigen -Isolating at least one cell from a mouse producing a chimeric mouse-human antibody, culturing at least one cell producing a reverse-chimeric mouse-human antibody specific for an antigen, and obtaining the antibody Includes.

In one embodiment, the reverse-chimeric mouse-human antibody comprises a human heavy chain variable domain fused to a mouse or rat heavy chain constant gene and a human light chain variable domain fused to a mouse or rat or human light chain constant gene.

In one embodiment, the step of culturing at least one cell producing an antigen-specific reverse-chimeric rodent-human antibody is performed on at least one hybridoma cell produced from at least one cell isolated from a mouse. do.

In one aspect, a method of generating a complete human antibody specific for an antigen of interest is provided, the method comprising immunizing a mouse with an antigen as described herein, a reverse-chimeric rodent specific for the antigen- Isolating at least one cell from a mouse producing human antibody, generating at least one cell that produces a fully human antibody derived from a reverse-chimeric rodent-human antibody specific for an antigen, a fully human antibody Culturing at least one cell to be produced, and obtaining the intact human antibody.

In various embodiments, at least one cell isolated from a mouse producing a reverse-chimeric rodent-human antibody specific for an antigen is a splenocyte or a B cell.

In various embodiments, the antibody is a monoclonal antibody.

In various embodiments, immunization with an antigen of interest is performed with a protein, DNA, a combination of DNA and protein, or cells expressing the antigen.

In one aspect, there is provided the use of a mouse as described herein for constructing a nucleic acid sequence encoding an immunoglobulin variable region or fragment thereof. In one embodiment, the nucleic acid sequence is used to construct a human antibody or antigen-binding fragment thereof. In one embodiment, the mouse is an antibody, multi-specific antibody (e.g., bi-specific antibody), scFv, bi-specific scFv, diabody, tribody, tetrabody, V-NAR, V _HH , V _L , F(ab), F(ab) ₂ , DVD (i.e., double variable domain antigen-binding protein), SVD (i.e., single variable domain antigen-binding protein), or bispecific T-cell engager (bispecific T-cell engager: BiTE).

In one aspect, there is provided a use of a mouse as described herein for introducing an ectopic ADAM6 sequence in a mouse lacking a functional endogenous mouse ADAM6 sequence, wherein the use comprises a mouse as described herein that is a functional endogenous mouse. And mating with a mouse lacking the ADAM6 sequence.

In one aspect, the use of genetic material from a mouse as described herein to construct a mouse with an ectopic ADAM6 sequence is provided. In one embodiment, the use includes nuclear transplantation using the nucleus of a mouse cell as described herein. In one embodiment, the use comprises the step of cloning a cell of a mouse as described herein for producing an animal derived from the cell. In one embodiment, the use includes using a mouse sperm or egg as described herein in the process of making a mouse containing an ectopic ADAM6 sequence.

In one aspect, a method of constructing a male mouse of childbearing potential comprising a modified immunoglobulin heavy chain site is provided, wherein the method comprises a first mouse germ cell comprising the modification of an endogenous immunoglobulin heavy chain site, and functional ADAM6 in a male mouse. Fertilizing with a second mouse germ cell comprising a gene or an ortholog or homolog or fragment thereof; Forming a fertilized cell; Developing the fertilized cell into an embryo; And fertilizing the embryo in the surrogate to obtain a mouse.

In one embodiment, fertilization is achieved by mating male and female mice. In one embodiment, the female mouse comprises the ADAM6 gene or an ortholog or homolog or fragment thereof. In one embodiment, the male mouse contains the ADAM6 gene or an ortholog or homolog or fragment thereof.

In one aspect, encoding a mouse ADAM6 protein or an ortholog or homolog thereof, or a functional fragment of the corresponding ADAM6 protein, for restoring or augmenting the fertility of a mouse with a genome comprising a modification of an immunoglobulin heavy chain site. The use of nucleic acid sequences is provided, wherein the modification reduces or eliminates endogenous ADAM6 function.

In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at an ectopic location. In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at the endogenous immunoglobulin site. In a specific embodiment, the endogenous immunoglobulin site is a heavy chain site. In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at a location other than the endogenous immunoglobulin site.

In one aspect, for the manufacture of a drug (eg , antigen-binding protein), or for the production of a sequence encoding a variable sequence of a drug (eg, antigen-binding protein), the present invention for the treatment of a human disease or disorder Use of a mouse as described herein is provided.

In one aspect, a genetically modified mouse cell is provided, wherein the cell makes it impossible to express a heavy chain comprising a rearranged endogenous immunoglobulin heavy chain gene segment, and the cell encodes a mouse ADAM6 protein or a functional fragment thereof. It contains a functional ADAM6 gene. In one embodiment, the cell further comprises an insert of a human immunoglobulin gene segment. In a specific embodiment, the human immunoglobulin gene segment is a heavy chain gene segment operably linked to a mouse heavy chain constant region encoding a functional heavy chain of an antibody comprising a human variable region upon rearrangement.

Provided are nucleic acid constructs for modifying non-human animals, as well as genetically modified non-human animals, embryos, cells, tissues, and methods and compositions for making and using them. Animals and cells are provided that produce a lambda (λ) variable region (human or non-human) in the context of a kappa (κ) light chain, wherein the animal and cell eliminate the activity of the ADAM6 protein or its homolog or ortholog. Or a modification of the heavy chain immunoglobulin site to reduce or reduce, wherein the animal further comprises a genetic modification that fully or partially restores ADAM6 activity (or its homolog or ortholog activity). A mouse is provided that expresses a human λ variable domain that is fertile and is cognate with a human heavy chain variable domain, wherein a λ or κ constant region and a contiguous human λ variable domain are expressed in the mouse, and in various embodiments, a λ or κ variable The region is an endogenous (eg, mouse or rat) constant region. Also provided are mice and cells that produce a human λ variable region in the context of a κ or λ light chain, eg, from an endogenous mouse light chain locus. Also provided is a method for producing an antibody comprising a lambda variable region. A method of selecting a heavy chain that is expressed with a cognate lambda variable region is provided.

Chimeric and human antigen-binding proteins (e.g., antibodies), comprising somatically mutated variable regions, including antibodies with light chains comprising variable domains derived from human Vλ and human Jλ gene segments fused to mouse light chain constant domains. , And a nucleic acid encoding them is provided.

In one aspect, a mouse is provided that expresses a human λ variable region sequence on a light chain comprising a mouse constant region. In one aspect, a mouse is provided that expresses a human λ variable region sequence on a light chain comprising a κ constant region. In one aspect, a mouse is provided that expresses a light chain comprising a human λ variable region sequence from an endogenous mouse light chain locus. In one aspect, there is provided a mouse comprising a rearranged light chain gene comprising a human λ variable sequence linked to a mouse constant region sequence; In one embodiment, the mouse constant region sequence is a λ constant sequence; In one embodiment, the mouse constant region sequence is a κ constant sequence.

In one aspect, a genetically modified mouse is provided, wherein the mouse comprises an unrearranged human λ light chain variable gene segment (hVλ) and a human λ splicing gene segment (hJλ). In one embodiment, the unrearranged hVλ and hJλ are at the mouse light chain locus. In one embodiment, the unrearranged hVλ and the unrearranged hJλ are on the transgene and are also operably linked to a human or mouse constant region sequence. In one embodiment, the unrearranged hVλ and the unrearranged hJλ are on the episome. In one embodiment, a mouse can produce an immunoglobulin comprising a mouse light chain constant region (C _L ) nucleic acid sequence, an unrearranged hVλ sequence, and a light chain derived from the hJλ sequence. Methods and compositions for making and using genetically modified mice are also provided. An antibody is provided, wherein the antibody comprises (a) a human heavy chain variable domain fused to a mouse heavy chain constant region (hV _H ) and (b) a human V _{L fused to a mouse C L} domain; At this time, one or more of the variable domains include somatic mutations in one or more of the variable domains in the mouse of the present invention, for example, during antibody or immune cell selection. In one embodiment, the unrearranged hVλ and the unrearranged hJλ are operably linked with a human or mouse κ constant region (Cκ). In one embodiment, the unrearranged hVλ and the unrearranged hJλ are operably linked with a human or mouse λ constant region (Cλ).

In one aspect, provided is a mouse comprising a human λ light chain variable region sequence at an endogenous mouse light chain site in the germline, wherein the human lambda variable region sequence is expressed in a light chain comprising a mouse immunoglobulin constant region gene sequence.

In one embodiment, the endogenous mouse light chain site is a λ site. In one embodiment, the endogenous mouse light chain site is a κ site.

In one embodiment, the mouse lacks an endogenous light chain variable sequence at the endogenous mouse light chain locus. In one embodiment, all or substantially all of the endogenous mouse light chain variable region gene segment is replaced with one or more human λ variable region gene segments.

In one embodiment, the human λ light chain variable region sequence comprises a human Jλ sequence. In one embodiment, the human Jλ sequence is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof.

In one embodiment, the human λ light chain variable region sequence comprises a fragment of cluster A of a human light chain locus. In a specific embodiment, the fragment of cluster A of the human λ light chain locus extends from Vλ3-27 through hVλ3-1.

In one embodiment, the human λ light chain variable region sequence comprises a fragment of cluster B of a human light chain locus. In a specific embodiment, the fragment of cluster B of the human λ light chain locus extends from hVλ5-52 through hVλ1-40.

In one embodiment, the human λ light chain variable region sequence comprises a fragment of genomic cluster A and a fragment of genomic cluster B. In one embodiment, the human λ light chain variable region sequence comprises at least one gene segment of cluster A and at least one gene segment of cluster B.

In one embodiment, at least 10% of the light chain naive repotri in mice are derived from at least two hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45 and 9-49. In one embodiment, at least 20% of the light chain naive repertoire of the mouse is derived from at least three hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45 and 9-49. In one embodiment, at least 30% of the light chain naive repertoire of the mouse is derived from at least four hVλ gene segments selected from 2-8, 2-23, 1-40, 5-45 and 9-49.

In one aspect, a mouse expressing an immunoglobulin light chain comprising a human λ variable sequence fused with a mouse constant region is provided, wherein the mouse exhibits a κ use to λ use ratio of about 1:1.

In one embodiment, the immunoglobulin light chain is expressed from an endogenous mouse light chain locus.

In one aspect, a mouse is provided comprising a λ light chain variable region sequence (Vλ) and at least one J sequence (J), contiguous with a mouse κ light chain constant region sequence.

In one embodiment, the mouse lacks a functional mouse VK and/or mouse JK gene segment.

In one embodiment, Vλ is human Vλ (hVλ) and J is human Jλ (hJλ). In one embodiment, hVλ and hJλ are unrearranged gene segments.

In one embodiment, the mouse comprises a plurality of unrearranged hVλ gene segments and at least one hJλ gene segment. In specific embodiments, the plurality of unrearranged hVλ gene segments are at least 12 gene segments, at least 28 gene segments, or at least 40 gene segments.

In one embodiment, the at least one hJλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof.

In one embodiment, the endogenous mouse light chain locus is wholly or partially deleted.

In one embodiment, the mouse light chain constant region sequence is at an endogenous mouse light chain site.

In one embodiment, about 10% to about 45% of mouse B cells express an antibody comprising a light chain comprising a human λ light chain variable (Vλ) domain and a mouse κ light chain constant (CK) domain.

In one embodiment, the human λ variable domain is 3-1/1, 3-1/7, 4-3/1, 4-3/7, 2-8/1, 3-9/1, 3-10 /1, 3-10/3, 3-10/7, 2-14/1, 3-19/1, 2-23/1, 3-25/1, 1-40/1, 1-40/2 , 1-40/3, 1-40/7, 7-43/1, 7-43/3, 1-44/1, 1-44/7, 5-45/1, 5-45/2, 5 -45/7, 7-46/1, 7-46/2, 7-46/7, 9-49/1, 9-49/2, 9-49/7 and 1-51/1 It is derived from a selected rearranged hVλ/hJλ sequence.

In one embodiment, the mouse further comprises a human Vκ-Jκ intergenic region from a human κ light chain locus, wherein the human Vκ-Jκ intergenic region is contiguous with the Vλ sequence and the J sequence. In a specific embodiment, the human Vκ-Jκ intergenic region is positioned between the Vλ sequence and the J sequence.

In one aspect, (a) at least 12 to at least 40 unrearranged human λ light chain variable region gene segments and at least one human Jλ gene segment at the endogenous mouse light chain locus; (b) a mouse comprising a human VK-JK intergenic sequence positioned between at least 12 to at least 40 human light chain variable region gene segments and at least one human Jλ sequence is provided; Here, the mouse expresses an antibody comprising a human Vλ domain and a light chain comprising a mouse Cκ domain.

In one aspect, a mouse expressing an antibody comprising a light chain comprising a λ variable sequence and a κ constant sequence is provided.

In one embodiment, the mice exhibit a ratio of κ usage to λ usage of about 1:1.

In one embodiment, the population of immature B cells obtained from the bone marrow of a mouse exhibits a ratio of κ usage to λ usage of about 1:1.

In one aspect, a genetically modified mouse is provided, wherein the mouse comprises an unrearranged immunoglobulin Vλ and Jλ gene segment operably linked to a mouse light chain site comprising a mouse _{C L gene.}

In one embodiment, the Vλ and/or Jλ gene segment is a human gene segment. In one embodiment, the Vλ and/or Jλ gene segment is a mouse gene segment, and C _L is a mouse Cκ.

In one embodiment, the endogenous mouse light chain locus is a κ light chain locus. In one embodiment, the endogenous mouse light chain locus is a λ light chain locus.

In one embodiment, the unrearranged Vλ and Jλ gene segments are at the endogenous mouse light chain site.

In one embodiment, the unrearranged immunoglobulin Vλ and Jλ gene segments are on a transgene.

In one embodiment, the mouse is further subjected to replacement of one or more heavy chain V, D, and/or J gene segments with one or more human V, D, and/or J gene segments at the endogenous mouse heavy chain immunoglobulin site. Includes.

In one embodiment, the mouse comprises an unrearranged immunoglobulin Vλ and Jλ gene segment at an endogenous mouse κ light chain site comprising a mouse Cκ gene.

In one embodiment, the mouse comprises a human immunoglobulin λ light chain variable gene segment (Vλ) and a λ splicing gene segment (Jλ) that are not rearranged in an endogenous mouse λ light chain site comprising a mouse Cλ gene.

In one embodiment, the light chain variable locus (“V _L locus”) comprises at least one human Vλ(hVλ) gene segment. In one embodiment, the V _L site comprises at least one human Jλ (hJλ) gene segment. In other embodiments, the V _L site comprises up to 4 hJλ gene segments. In one embodiment, the V _L site comprises a contiguous sequence comprising human λ and human κ genomic sequences.

In one embodiment, the κ light chain variable locus (“κ site”) comprises at least one human Vλ (hVλ) gene segment. In one embodiment, the κ site comprises at least one human Jλ (hJλ) gene segment. In one embodiment, the κ site comprises up to 4 hJλ gene segments. In one embodiment, the κ site comprises at least one hVλ and at least one hJλ, lacks or substantially lacks a functional Vκ region gene segment, and lacks or substantially lacks a functional Jκ region gene segment. In one embodiment, the mouse does not contain a functional Vκ region gene segment. In one embodiment, the mouse does not contain a functional Jκ region gene segment.

In one embodiment, the λ light chain variable locus (“λ site”) comprises at least one hVλ gene segment. In one embodiment, the λ site comprises at least one human Jλ (hJλ) gene segment. In other embodiments, the λ site comprises up to 4 hJλ gene segments.

In one embodiment, the V _L site includes a plurality of hVλ. In one embodiment, the plurality of hVλ is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about Vλ use observed in humans. It is chosen to result in the expression of a λ light chain variable region repertoire reflecting at least 90%. In one embodiment, the V _L site comprises the gene segments hVλ 1-40, 1-44, 2-8, 2-14, 3-21, and combinations thereof.

In one embodiment, hVλ includes 3-1, 4-3, 2-8, 3-9, 3-10, 2-11 and 3-12. In a specific embodiment, the V _L site comprises a contiguous sequence of human λ light chain sites from Vλ3-12 to Vλ3-1. In one embodiment, the V _L site comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hVλ. In a specific embodiment, hVλ includes 3-1, 4-3, 2-8, 3-9, 3-10, 2-11 and 3-12. In a specific embodiment, the V _L site comprises a contiguous sequence of human λ sites from Vλ3-12 to Vλ3-1. In one embodiment, the V _L site is at an endogenous κ site. In a specific embodiment, the V _L site is at the endogenous κ site and the endogenous λ light chain site is partially or completely deleted. In one embodiment, the V _L site is in the endogenous λ site. In a specific embodiment, the V _L site is at the endogenous λ site, and the endogenous κ site is partially or completely deleted.

In one embodiment, the V _L site comprises 13 to 28 or more hVλ. In a specific embodiment, hVλ includes 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, and 3-27. In a specific embodiment, the κ site comprises a contiguous sequence of the human λ site from Vλ3-27 to Vλ3-1. In one embodiment, the V _L site is at an endogenous κ site. In a specific embodiment, the V _L site is at the endogenous κ site, and the endogenous λ light chain site is partially or completely deleted. In another embodiment, the V _L site is at the endogenous λ site. In a specific embodiment, the V _L site is at the endogenous λ site, and the endogenous κ site is partially or completely deleted.

In one embodiment, the V _L site comprises 29 to 40 hVλ. In a specific embodiment, the κ site comprises a contiguous sequence of human λ sites from Vλ3-29 to Vλ3-1 and a contiguous sequence of human λ sites from Vλ5-52 to Vλ1-40. In a specific embodiment, all or substantially all of the sequence between hVλ1-40 and hVλ3-29 in the genetically modified mouse is in nature downstream of the hVλ1-40 gene segment (downstream of the 3'untranslated portion). Essentially a human λ sequence of approximately 959 bp found (e.g., in a human population), a restriction enzyme site (e.g., PI-SceI), followed by a human λ sequence of approximately 3,431 bp upstream of the hVλ3-29 gene segment found in nature. It consists of. In one embodiment, the V _L site is at an endogenous mouse κ site. In a specific embodiment, the V _L site is at the endogenous mouse κ site, and the endogenous mouse λ light chain site is partially or completely deleted. In another embodiment, the V _L site is at the endogenous mouse λ site. In a specific embodiment, the V _L site is at the endogenous mouse λ site, and the endogenous mouse κ site is partially or completely deleted.

In one embodiment, the V _L site comprises at least one hJλ. In one embodiment, the V _L site includes a plurality of hJλ. In one embodiment, the V _L site comprises at least 2, 3, 4, 5, 6 or 7 hJλ. In a specific embodiment, the V _L site includes 4 hJλ. In a specific embodiment, the four hJλ are hJλ1, hJλ2, hJλ3 and hJλ7. In one embodiment, the V _L site is a κ site. In a specific embodiment, the V _L site is at the endogenous κ site, and the endogenous λ light chain site is partially or completely deleted. In one embodiment, the V _L site contains 1 hJλ. In a specific embodiment, one hJλ is hJλ1. In one embodiment, the V _L site is at an endogenous κ site. In a specific embodiment, the V _L site is at the endogenous κ site, and the endogenous λ light chain site is partially or completely deleted. In another embodiment, the V _L site is at the endogenous λ site. In a specific embodiment, the V _L site is at the endogenous λ site, and the endogenous κ site is partially or completely deleted.

In one embodiment, the V _L site comprises at least one hVλ, at least one hJλ, and a mouse Cκ gene. In one embodiment, the V _L site comprises at least one hVλ, at least one hJλ, and a mouse Cλ gene. In a specific embodiment, the mouse Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, 96%, 97%, 98% or at least 99% identical to mouse Cλ2.

In one embodiment, the mouse comprises replacement of an endogenous mouse Vκ gene segment with one or more hVλ gene segments at the endogenous mouse κ site, wherein the hVλ gene segment is operably linked to the endogenous mouse Cκ region gene, so that the mouse is The human Vλ gene segment is rearranged and the reverse chimeric immunoglobulin light chain comprising the human Vλ domain and mouse Cκ is expressed. In one embodiment, 90-100% of the unrearranged mouse VK gene segments are replaced with at least one unrearranged hVλ gene segment. In a specific embodiment, all or substantially all of the endogenous mouse Vκ gene segment is replaced with at least one unrearranged hVλ gene segment. In one embodiment, the replacement is done with at least 12, at least 28 or at least 40 unrearranged hVλ gene segments. In one embodiment, the replacement is at least 7 functional unrearranged hVλ gene segments. , At least 16 functional unrearranged hVλ gene segments, or at least 27 functional unrearranged hVλ gene segments. In one embodiment, the mouse comprises replacement of all mouse JK gene segments with at least one unrearranged hJλ gene segment. In one embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ4, Jλ5, Jλ6, Jλ7, and combinations thereof. In a specific embodiment, the one or more hVλ gene segments are 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, 3-12, 2-14, 3-16, 2- 18, 3-19, 3-21, 3-22, 2-23, 3-25, 3-27, 1-40, 7-43, 1-44, 5-45, 7-46, 1-47, 5-48, 9-49, 1-50, 1-51, 5-52 hVλ gene segments, and combinations thereof. In a specific embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof.

In one embodiment, the mouse comprises replacement of an endogenous mouse Vλ gene segment at the endogenous mouse λ site with one or more human Vλ gene segments at the endogenous mouse λ site, wherein the hVλ gene segment operates on a mouse Cλ region gene. Being possibly linked, the mouse rearranges the hVλ gene segment and also contains an inverted chimeric reverse globulin light chain comprising the hVλ domain and mouse Cλ. In a specific embodiment, the mouse Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2. In one embodiment, 90-100% of the unrearranged mouse Vλ gene segments are replaced with at least one unrearranged hVλ gene segment. In a specific embodiment, all or substantially all of the endogenous mouse Vλ gene segment is replaced with at least one unrearranged hVλ gene segment. In one embodiment, the replacement is done with at least 12, at least 28, or at least 40 unrearranged hVλ gene segments. In one embodiment, the replacement is at least 7 functional unrearranged hVλ gene segments. At least 16 functional unrearranged hVλ gene segments or at least 27 functional unrearranged hVλ gene segments. In one embodiment, the mouse comprises replacement of all mouse Jλ gene segments with at least one unrearranged hJλ gene segment. In one embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ4, Jλ5, Jλ6, Jλ7, and combinations thereof. In a specific embodiment, the one or more hVλ gene segments are 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, 3-12, 2-14, 3-16, 2- 18, 3-19, 3-21, 3-22, 2-23, 3-25, 3-27, 1-40, 7-43, 1-44, 5-45, 7-46, 1-47, 5-48, 9-49, 1-50, 1-51, 5-52 hVλ gene segments, and combinations thereof. In a specific embodiment, the at least one unrearranged hJλ gene segment is selected from Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof.

In one aspect, a genetically modified mouse is provided comprising a human Vκ-Jκ intergenic region sequence located at an endogenous mouse κ light chain site.

In one embodiment, the human Vκ-Jκ intergenic region sequence is at the endogenous κ light chain site of a mouse including the hVλ and hJλ gene segments, and the human Vκ-Jκ intergenic region sequence is between the hVλ gene segment and the hJλ gene segment. Is placed. In a specific embodiment, the hVλ and hJλ gene segments can be recombined into a functional human λ light chain variable domain in a mouse.

In one embodiment, a mouse is provided comprising a plurality of hVλ and one or more hJλ, and the human Vκ-Jκ intergenic region sequence is, with respect to transcription, a proximal or 3'downstream of most of the hVλ sequence and a first It is positioned upstream or 5'of the hJλ sequence.

In one embodiment, the human Vκ-Jκ intergenic region is about 130 bp downstream or 3'of the human Vκ4-1 gene segment, and about 130 bp downstream of the 3'untranslated region of the human Vκ4-1 gene segment. It is an arranged region and spans about 600 bp upstream or 5'of the human JK1 gene segment. In a specific embodiment, the human VK-JK intergenic region is about 22.8 kb in size. In one embodiment, the Vκ-Jκ intergenic region is a human Vκ-Jκ intergenic region extending from the end of the 3'untranslated region of the human Vκ4-1 gene segment to about 600 bp upstream of the human Jκ1 gene segment and about At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least about 95% are the same. In one embodiment, the Vκ-Jκ intergenic region comprises SEQ ID NO: 158. In a specific embodiment, the Vκ-Jκ intergenic region comprises a functional fragment of SEQ ID NO: 158. In a specific embodiment, the Vκ-Jκ intergenic region is SEQ ID NO: 158.

In one aspect, a non-human animal, non-human cell (e.g., ES cell or pluripotent cell), non-human embryo or non-human tissue comprising the mentioned human Vκ-Jκ intergenic region sequence is provided, , Where the intergenic region sequence is ectopic. In a specific embodiment, the ectopic sequence is placed at a humanized endogenous non-human immunoglobulin site. In one embodiment, the non-human animal is selected from mice, rats, hamsters, goats, cattle, sheep and non-human primates.

In one aspect, an isolated nucleic acid construct comprising the mentioned human VK-JK intergenic region sequence is provided. In one embodiment, the nucleic acid construct comprises a targeting arm that targets a human VK-JK intergenic region sequence to a mouse light chain site. In a specific embodiment, the mouse light chain site is a κ site. In a specific embodiment, the targeting section targets the human Vκ-Jκ intergenic region to a modified endogenous mouse κ site, wherein the targeting is to a position between the Vλ sequence and the hJλ sequence.

In one aspect, a genetically modified mouse is provided, wherein the mouse comprises no more than two light chain alleles, and the light chain allele is not rearranged to an endogenous mouse light chain site comprising _{(a) a mouse C L gene.} Non-immunoglobulin human Vλ and Jλ gene segments; And (b) _{immunoglobulin V L} and J _L gene segments that are not rearranged at the endogenous mouse light chain site containing the _{mouse C L gene.}

In one embodiment, the endogenous mouse light chain site is a κ site. In another embodiment, the endogenous mouse light chain site is a λ site.

In one embodiment, no more than two light chain alleles are selected from a κ allele and a λ allele, two κ alleles, and two λ alleles. In a specific embodiment, one of the two light chain alleles is a λ allele comprising the Cλ2 gene.

In one embodiment, the mouse comprises one functional immunoglobulin light chain locus and one non-functional light chain locus, wherein the functional light chain locus is an immunoglobulin human that is not rearranged to an endogenous mouse κ light chain locus comprising a mouse Cκ gene. It includes the Vλ and Jλ gene segments.

In one embodiment, the mouse comprises one functional immunoglobulin light chain locus and one non-functional light chain locus, wherein the functional light chain locus is an immunoglobulin human that is not rearranged to an endogenous mouse λ light chain locus comprising a mouse Cλ gene. It includes the Vλ and Jλ gene segments. In one embodiment, the Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to the mouse Cλ2.

In one embodiment, the mouse further comprises at least one immunoglobulin heavy chain allele. In one embodiment, the at least one immunoglobulin heavy chain allele comprises a human V _H gene segment, a human D _H gene segment, and a human J _H gene segment comprising a human heavy chain gene expressing a human/mouse heavy chain. In a specific embodiment, the mouse contains two immunoglobulin heavy chain alleles, and the mouse expresses a human/mouse heavy chain.

In one embodiment, the mouse comprises a first light chain allele comprising an unrearranged hVκ and an unrearranged hJκ in an endogenous mouse κ site comprising an endogenous Cκ gene; And a second light chain allele comprising an unrearranged hVλ and an unrearranged hJλ at an endogenous mouse κ site comprising an endogenous Cκ gene. In a specific embodiment, the first and second light chain alleles are the only functional light chain alleles of a genetically modified mouse. In a specific embodiment, the mouse comprises a non-functional λ site. In one embodiment, the genetically modified mouse does not express a light chain comprising a λ constant region.

In one embodiment, the mouse comprises a first light chain allele comprising an unrearranged hVκ and an unrearranged hJκ at an endogenous mouse κ site comprising an endogenous Cκ gene; And a second light chain allele comprising an unrearranged hVλ and an unrearranged hJλ at an endogenous mouse λ site comprising an endogenous Cλ gene. In a specific embodiment, the first and second light chain alleles are the only functional light chain alleles of a genetically modified mouse. In one embodiment, the endogenous Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

In one embodiment, the mouse comprises 6 immunoglobulin alleles, wherein the first allele comprises an unrearranged immunoglobulin Vλ and Jλ gene segment at an endogenous mouse κ light chain site comprising a mouse Cκ gene, and , The second allele contains unrearranged immunoglobulin Vκ and Jκ gene segments in the endogenous mouse κ light chain site containing the mouse Cκ gene, and the third allele is in the endogenous mouse λ light chain site containing the mouse Cλ gene. _{V H} and D _H and J _H genes that are not rearranged in endogenous mouse heavy chain loci containing unrearranged immunoglobulin Vλ and Jλ gene segments, and the fourth and fifth alleles are each independently a mouse heavy chain gene. Segment, and the sixth allele is (a) an unrearranged immunoglobulin Vλ and Jλ gene segment at the endogenous mouse λ light chain site containing the mouse Cλ gene, (b) a non-functional λ site, or (c) Includes full or partial deletion of the λ position.

In one embodiment, the first allele comprises unrearranged hVλ and hJλ. In one embodiment, the second allele comprises unrearranged hVκ and hJκ. In one embodiment, the third allele comprises unrearranged hVλ and hJλ. In one embodiment, the fourth and fifth alleles each independently comprise unrearranged hV _H and hD _H and hJ _H. In one embodiment, the sixth allele comprises a wholly or partially deleted endogenous mouse λ locus.

In one embodiment, the mouse comprises 6 immunoglobulin alleles, wherein the first allele comprises an unrearranged immunoglobulin Vλ and Jλ gene segment at the endogenous mouse λ light chain site comprising the mouse Cλ gene, and , The second allele contains unrearranged immunoglobulin Vλ and Jλ gene segments in the endogenous mouse λ light chain locus containing the mouse Cλ gene, and the third allele is the endogenous mouse κ light chain locus containing the mouse Cκ gene. _{V H} and D _H and J _H unrearranged to endogenous mouse heavy chain loci containing immunoglobulin Vκ and Jκ gene segments that are not rearranged in, and the fourth and fifth alleles are each independently a mouse heavy chain gene. A gene segment, and the sixth allele is (a) an unrearranged immunoglobulin Vκ and Jκ gene segment at the endogenous mouse κ light chain site containing the mouse Cκ gene, (b) a non-functional κ site, or (c ) contains the deletion of one or more elements of the κ site.

In one embodiment, the first allele comprises an unrearranged hVλ and hJλ gene segments. In one embodiment, the second allele comprises an unrearranged hVλ and hJλ gene segments. In one embodiment, the third allele comprises an unrearranged hVK and hJK gene segments. In one embodiment, the fourth and fifth alleles each comprise independently unrearranged hV _H and hD _H and hJ _H gene segments. In one embodiment, the sixth allele comprises a functionally silent endogenous mouse κ site

In one embodiment, the genetically modified mouse comprises a B cell comprising a rearranged antibody gene comprising a rearranged hVλ domain operably linked to a _{mouse C L domain.} In one embodiment, the mouse C _L domain is selected from a mouse CK and a mouse Cλ domain. In a specific embodiment, the mouse Cλ domain is derived from the Cλ2 gene. In a specific embodiment, the mouse Cλ domain is derived from a Cλ domain that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

In one aspect, a genetically modified mouse expressing a Vλ region on _{C L, which is Cκ, is provided.} In one aspect, a genetically modified mouse expressing an hVλ region on a _{C L} selected from human Cκ, human Cλ or mouse Cκ is provided. In one aspect, a genetically modified mouse expressing an hVλ region on mouse Cκ is provided.

In one embodiment, about 10-50% of the splenocytes of the mouse are B cells (i.e., CD19-positive), or about 9-28% of them are immunoglobulin light chains comprising an hVλ domain fused to a mouse Cκ domain. Express.

In a specific embodiment, about 23-34% of the splenocytes of the mouse are B cells (i.e., CD19-positive), or about 9-11% of them are immunoglobulins comprising hVλ domains fused to a mouse Cκ domain. Express the light chain.

In a specific embodiment, about 19-31% of the splenocytes of the mouse are B cells (i.e., CD19-positive), or about 9-17% of them are immunoglobulins comprising hVλ domains fused to a mouse Cκ domain. Express the light chain.

In specific embodiments, about 21-38% of the splenocytes of the mouse are B cells (i.e., CD19-positive), or about 24-27% of them are immunoglobulins comprising hVλ domains fused to a mouse Cκ domain. Express the light chain.

In a specific embodiment, about 10-14% of the splenocytes of the mouse are B cells (i.e., CD19-positive), or about 9-13% of them are immunoglobulins comprising an hVλ domain fused to a mouse Cκ domain. Express the light chain.

In a specific embodiment, about 31-48% of the splenocytes of the mouse are B cells (i.e., CD19-positive), or about 15-21% of them are immunoglobulins comprising hVλ domains fused to a mouse Cκ domain. Express the light chain. In a specific embodiment, about 30-38% of the splenocytes of the mouse are B cells (i.e., CD19-positive), or about 33-48% of them are immunoglobulins comprising hVλ domains fused to a mouse Cκ domain. Express the light chain.

In one embodiment, about 52-70% of the bone marrow of the mouse is B cells (i.e., CD19-positive), or of immature B cells (i.e., CD19-positive/B220-intermediate positive/IgM-positive) among them. About 31-47% express an immunoglobulin light chain comprising an hVλ domain fused to a mouse CK domain.

In one embodiment, about 60% of the bone marrow of a mouse is B cells (i.e., CD19-positive), or about 38.3 of immature B cells (i.e., CD19-positive/B220-intermediate positive/IgM-positive) among them. % Expresses an immunoglobulin light chain comprising an hVλ domain fused to a mouse Cκ domain.

In one embodiment, the mouse expresses an antibody comprising a light chain comprising a variable domain derived from a human V and human J gene segment and a constant domain derived from a mouse constant region gene. In one embodiment, the mouse constant region gene is a Cκ gene. In another embodiment, the mouse constant region gene is a Cλ gene. In a specific embodiment, the Cλ region is Cλ2. In a specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2. In a specific embodiment, the antibody further comprises a heavy chain comprising a variable domain derived from human V, human D and human J gene segments and a heavy chain constant domain derived from a mouse heavy chain constant region gene. In one embodiment, the mouse heavy chain constant region gene comprises a hinge-CH2-CH3 sequence of a heavy chain constant domain. In another embodiment, the mouse heavy chain constant region gene comprises a CH1-hinge-CH2-CH3 sequence of a heavy chain constant domain. In another embodiment, the mouse heavy chain constant region gene comprises the CH1-CH2-CH3-CH4 sequence of a heavy chain constant domain. In another embodiment, the mouse heavy chain constant region gene comprises the CH2-CH3-CH4 sequence of a heavy chain constant domain.

In one embodiment, the mouse expresses an antibody comprising a rearranged human Vλ-Jλ sequence and a light chain comprising a mouse CK sequence. In one embodiment, the rearranged human Vλ-Jλ sequence is 3-1, 4-3, 2-8, 3-9, 3-10, 2-14, 3-19, 2-23, 3-25 , 1-40, 7-43, 1-44, 5-45, 7-46, 1-47, 9-49 and 1-51 gene segments. In one embodiment, the rearranged human Vλ-Jλ sequence is derived from a rearrangement of an hJλ gene segment selected from the Jλ1, Jλ2, Jλ3 and Jλ7 gene segments.

In one embodiment, the mouse is 3-1/1, 3-1/7, 4-3/1, 4-3/7, 2-8/1, 3-9/1, 3-10/1, 3-10/3, 3-10/7, 2-14/1, 3-19/1, 2-23/1, 3-25/1, 1-40/1, 1-40/2, 1- 40/3, 1-40/7, 7-43/1, 7-43/3, 1-44/1, 1-44/7, 5-45/1, 5-45/2, 5-45/ Human Vλ/Jλ sequence selected from 7, 7-46/1, 7-46/2, 7-46/7, 9-49/1, 9-49/2, 9-49/7 and 1-51/1 It expresses an antibody comprising a light chain comprising a rearranged immunoglobulin λ light chain variable region comprising a. In a specific embodiment, the B cells express an antibody comprising a human immunoglobulin heavy chain variable domain fused to a mouse heavy chain constant domain and a human immunoglobulin λ light chain variable domain fused to a mouse κ light chain constant domain.

In one aspect, (a) a heavy chain comprising a heavy chain variable domain derived from an unrearranged human heavy chain variable region gene segment, wherein the heavy chain variable domain is fused to a _{mouse heavy chain constant (C H) region;} And (b) a light chain comprising a light chain variable domain derived from unrearranged hVλ and hJλ, wherein the light chain variable domain is fused to the _{mouse C L region.}

In one embodiment, the mouse is (i) all or substantially all of the functional endogenous mouse V, D and J gene segments are replaced with all or substantially all functional human V, D and J gene segments, and mouse C _{A heavy chain locus comprising the H} gene, (ii) all or substantially all of the functional endogenous mouse Vκ and Jκ gene segments with replacement of all, substantially all or a plurality of, functional hVλ and hJλ gene segments, and the mouse Cλ gene A first κ light chain site comprising, (iii) replacement of all, substantially all or a plurality of, functional hVκ and hJκ gene segments of all or substantially all of the functional endogenous mouse Vκ and Jκ gene segments, and the mouse Cκ gene A second κ light chain site comprising In one embodiment, the mouse does not express an antibody comprising a Cλ region. In one embodiment, the mouse comprises a deletion of a Cλ gene and/or a Vλ and/or Jλ gene segment. In one embodiment, the mouse comprises a non-functional λ light chain site. In a specific embodiment, the λ light chain site is wholly or partially deleted.

In one embodiment, the mouse is (i) all or substantially all of the functional endogenous mouse V, D and J gene segments are replaced with all or substantially all functional human V, D and J gene segments, mouse C _H A heavy chain locus comprising a gene, (ii) replacement of all, substantially all or a plurality of, functional hVλ and hJλ gene segments of all or substantially all functional endogenous mouse Vλ and Jλ gene segments, and a mouse Cλ gene. A first λ light chain locus, (iii) replacement of all, substantially all or a plurality of, functional hVλ and hJλ gene segments of all or substantially all functional endogenous mouse Vλ and Jλ gene segments, and a mouse Cλ gene. Contains the second λ light chain site. In a specific embodiment, the mouse Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

In one embodiment, the mouse comprises a deletion of a Cκ gene and/or a Vκ and/or Jκ gene segment. In one embodiment, the mouse comprises a non-functional κ light chain site.

In one aspect, a genetically modified mouse expressing an antibody is provided, wherein 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more of the total IgG antibodies produced by the mouse , 40% or more, 60% or more, 70% or more, 80% or more, or 90% or more contain a λ-derived variable domain, and the mouse expresses an antibody comprising a κ-derived variable domain fused to the mouse Cκ region do. In a specific embodiment, about 15-40%, 20-40%, 25-40%, 30-40% or 35-40% of the total IgG antibodies produced by the mouse comprise a λ-derived variable domain.

In one embodiment, the λ-derived variable domain is derived from hVλ and hJλ. In one embodiment, the λ-derived variable domain is within a light chain comprising a mouse Cκ region. In a specific embodiment, the λ-derived variable region is in the light chain comprising the mouse Cλ region. In another specific embodiment, the Cλ region is a Cλ2 region. In one embodiment, the κ-derived variable domain is derived from hVκ and hJκ, and in a specific embodiment is in the light chain containing the mouse Cκ region.

In one aspect, an isolated DNA construct is provided comprising an upstream homology section and a downstream homology section, wherein the upstream and downstream homology sections target the construct to a mouse κ site, and the construct is It includes a functional non-rearranged hVλ segment and a functional non-rearranged hJλ segment, and a selection or marker sequence.

In one aspect, with respect to the direction of transcription from 5'to 3', the targeting section for targeting the mouse λ sequence upstream of mouse Vλ2, the selection cassette on the 5'and 3'sides of the recombinase recognition site, and 3 of the mouse Jλ2 An isolated DNA construct comprising a targeting moiety for targeting a mouse λ sequence in 'is provided. In one embodiment, the selection cassette is a Frt'ed Hyg-TK cassette. In one embodiment, the 3'targeting segment comprises mouse Cλ2, Jλ4, Cλ4, and mouse enhancer 2.4.

In one aspect, with respect to the transcription direction from 5'to 3', a targeting moiety targeting the mouse λ site at 5'with respect to Vλ1, a selection cassette on the 5'and 3'sides of the recombinase recognition site, and mouse Cλ1 An isolated DNA construct comprising a 3'targeting section targeting the mouse λ sequence at 3'is provided. In one embodiment, the selection cassette is a loxed neomycin cassette. In one embodiment, the 3'targeting section comprises a mouse λ 3'enhancer and a mouse λ 3'enhancer 3.1.

In one aspect, with respect to the direction of transcription from 5'to 3', a targeting section for targeting the mouse λ site at 5'with respect to Vλ2, a selection cassette on the 5'and 3'sides of the recombinase recognition site, and a mouse An isolated DNA construct comprising a 3'targeting section to target the mouse λ sequence at 3'for Jλ2 and 5'for mouse Cλ2 is provided. In one embodiment, the selection cassette is an Frt-attached hydromycin-TK cassette. In one embodiment, the 3'targeting segment comprises a mouse Cλ2-Jλ4-Cλ4 gene segment and a mouse λ enhancer 2.4.

In one aspect, with respect to the transcription direction from 5'to 3', a targeting moiety targeting the mouse λ site at 5'with respect to Vλ2, a selection cassette on the 5'and 3'sides of the recombinase recognition site, hVλ3-12 An isolated DNA construct comprising a human genomic fragment comprising a contiguous region of a human λ light chain site downstream from the end of hJλ1, and a 3'targeting segment targeting the mouse λ sequence at 3'with respect to mouse Jλ2. Is provided. In one embodiment, the selection cassette is a neomycin cassette with Frt. In one embodiment, the 3'targeting segment comprises a mouse Cλ2-Jλ4-Cλ4 gene segment and a mouse λ enhancer 2.4.

In one aspect, an isolated DNA construct comprising a contiguous region of a human λ light chain site downstream from hVλ3-12 to hJλ1 is provided.

In one aspect, with respect to the transcription direction from 5'to 3', a targeting moiety targeting the mouse λ site at 5'with respect to Vλ2, a selection cassette on the 5'and 3'sides of the recombinase recognition site, and hVλ3-27 An isolated DNA construct is provided comprising a human genomic fragment comprising a contiguous region of a human λ light chain site downstream from to the end of hVλ2-8. In one embodiment, the selection cassette is a hygromycin cassette with Frt. In one embodiment, the human genomic fragment comprises a 3'targeting segment. In a specific embodiment, the 3'targeting segment comprises about 53 kb of a human λ light chain site downstream from hVλ3-12 to the end of hVλ2-8.

In one aspect, an isolated DNA construct is provided comprising a contiguous region of a human λ light chain site downstream from hVλ3-27 to the end of hVλ3-12.

In one aspect, with respect to the transcription direction from 5'to 3', a targeting moiety targeting the mouse λ site at 5'with respect to Vλ2, a selection cassette on the 5'and 3'sides of the recombinase recognition site, hVλ5-52 A first human genome fragment comprising a contiguous region of a human λ light chain site downstream from to the end of hVλ1-40, a restriction enzyme site, and a continuation of the human λ light chain site downstream from hVλ3-29 to the end of hVλ82K An isolated DNA construct comprising a second human genomic fragment comprising a region is provided. In one embodiment, the selection cassette is a neomycin cassette with Frt. In one embodiment, the restriction enzyme site is a site for homing endonuclease. In a specific embodiment, the homing endonuclease is PI-SceI. In one embodiment, the second human genomic fragment is a 3'targeting segment. In a specific embodiment, the 3'targeting segment comprises about 27 kb of a human λ light chain site downstream from hVλ3-29 to the end of hVλ82K.

In one aspect, an isolated DNA construct is provided comprising a contiguous region of a human λ light chain site downstream from hVλ5-52 to the end of hVλ1-40.

In one aspect, with respect to the direction of transcription from 5'to 3', with respect to the endogenous Vκ gene segment, a targeting moiety targeting the mouse κ site at 5', two juxtaposed recombinase recognition sites, juxtaposed recombination at 3' An isolated DNA construct is provided comprising a selection cassette to the enzyme recognition site, and a 3'targeting segment targeting the mouse κ sequence to 5'with respect to the κ light chain variable gene segment.

In one embodiment, the juxtaposed recombinase recognition sites are in opposite orientations with respect to each other. In a specific embodiment, the recombinase recognition sites are different. In another specific embodiment, the recombinase recognition site is a lox P site and a lox 511 site. In one embodiment, the selection cassette is a neomycin cassette.

In one aspect, with respect to the direction of transcription from 5'to 3', a targeting section targeting the mouse κ site at 5'with respect to the mouse Jκ gene segment, a selection cassette, a recombinase recognition site from 3'to the selection cassette, and An isolated DNA construct is provided comprising a 3'targeting section targeting a mouse κ sequence to a mouse κ intron enhancer at 3'and 5'with respect to the mouse Jκ gene segment. In one embodiment, the selection cassette is a hygromycin-TK cassette. In one embodiment, the recombinase recognition site is in the same orientation with respect to the selection cassette and transcription. In a specific embodiment, the recombinase recognition site is a lox P site.

In one aspect, with respect to the direction of transcription from 5'to 3', a first mouse genome fragment comprising a sequence 5'of an endogenous mouse Vκ gene segment, a first recombinase recognition site, a second recombinase recognition site, and An isolated DNA construct is provided comprising a second mouse genomic fragment comprising a sequence 3′ to the endogenous mouse JK gene segment and 5′ to the mouse κ intron enhancer.

In one aspect, a genetically modified mouse is provided, wherein the genetic modification comprises a modification having one or more of the above or the DNA constructs described herein.

In one aspect, the use of an isolated DNA construct for constructing a mouse as described herein is provided. In one aspect, provided is the use of an isolated DNA construct in a method as described herein to construct an antigen-binding protein.

In one aspect, a non-human stem cell comprising a targeting vector comprising a DNA construct as described above and herein is provided. In one aspect, a non-human stem cell is provided, wherein the non-human stem cell is derived from a mouse described herein.

In one embodiment, the non-human stem cell is an embryonic stem (ES) cell. In a specific embodiment, the ES cell is a mouse ES cell.

In one aspect, there is provided the use of a non-human stem cell as described herein for making a mouse as described herein. In one aspect, the use of a non-human stem cell as described herein for constructing an antigen-binding protein is provided.

In one aspect, a mouse embryo is provided, wherein the mouse embryo comprises a genetic modification as provided herein. In one embodiment, a host mouse embryo comprising a donor ES cell is provided, wherein the donor ES cell comprises a genetic modification as described herein. In one embodiment, the mouse embryo is a pre-lost embryo. In a specific embodiment, the pre-lost stage embryo is a 4-cell stage embryo or an 8-cell stage embryo. In another specific embodiment, the mouse embryo is a blastocyst.

In one aspect, there is provided the use of a mouse embryo as described herein for making a mouse as described herein. In one aspect, the use of a mouse embryo as described herein for making an antigen-binding protein is provided.

In one aspect, a non-human cell is provided, wherein the non-human cell comprises a rearranged immunoglobulin light chain gene sequence derived from a genetically modified mouse as described herein. In one embodiment, the cell is a B cell. In one embodiment, the cell is a hybridoma. In one embodiment, the cell encodes a somatic mutated immunoglobulin light chain variable domain and/or immunoglobulin heavy chain variable domain.

In one aspect, a non-human cell is provided, wherein the non-human cell comprises a rearranged immunoglobulin light chain gene sequence derived from a genetically modified mouse as described herein. In one embodiment, the cell is a B cell. In one embodiment, the cell is a hybridoma. In one embodiment, the cell encodes a somatic mutated immunoglobulin light chain variable domain and/or immunoglobulin heavy chain variable domain.

In one aspect, there is provided the use of a non-human cell as described herein for making a non-human animal as described herein. In one aspect, the use of a non-human cell as described herein for constructing an antigen-binding protein is provided. In one embodiment, the non-human animal is selected from mice, rats, hamsters, sheep, goats, cattle and non-human primates.

In one aspect, (a) a variable region derived from an hVλ gene segment and an hJλ gene segment; And (b) mouse B cells expressing an immunoglobulin light chain comprising a _{mouse C L gene.} In one embodiment, the mouse C _L gene is selected from Cκ and Cλ genes. In a specific embodiment, the Cλ gene is Cλ2. In a specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2. In one embodiment, the mouse B cells further express a cognate heavy chain comprising _{(c) a variable region derived from hV H} , hD _H and (d) an hJ _{H segment.} In one embodiment, the B cells do not contain a rearranged λ gene. In another embodiment, the B cells do not contain a rearranged κ gene.

In one aspect, a method of producing an antibody in a genetically modified non-human animal is provided, the method comprising: (a) exposing the genetically modified non-human animal to an antigen, wherein the animal is at least at the endogenous light chain site. Has a genome comprising one hVλ and at least one hJλ, and the endogenous light chain locus contains a non-human C _L gene); (b) developing an immune response to the antigen in the genetically modified animal; And, (c) isolating an antibody that specifically recognizes the antigen (b) from the animal or (b) isolating a cell comprising an immunoglobulin domain that specifically recognizes the antigen from the animal, wherein Antibodies include light chains derived from hVλ, hJλ and animal C _{L genes.} In a specific embodiment, the non-human C _L gene is a mouse CK gene. In one embodiment, the non-human animal is selected from mice, rats, hamsters, rabbits, sheep, goats, cattle, and non-human primates.

In one embodiment, a method for producing an antibody in a genetically modified non-human animal is provided, the method comprising: (a) exposing the genetically modified animal to an antigen, wherein the animal is at least one endogenous κ site. hVλ and a genome comprising at least one hJλ at the κ site, the κ site containing a non-human Cκ gene); (b) developing an immune response to the antigen in the genetically modified animal; And (c) isolating (b) an antibody that specifically recognizes the antigen from the animal or (b) isolating a cell comprising an immunoglobulin domain that specifically recognizes the antigen from the mouse, wherein Antibodies include light chains derived from hVλ, hJλ and non-human Cκ genes.

In one embodiment, the κ light chain constant gene is selected from a human Cκ gene and a mouse Cκ gene.

In one embodiment, a method of producing an antibody in a genetically modified non-human animal is provided, the method comprising: (a) exposing the genetically modified non-human animal to an antigen, wherein the animal is at the λ light chain site. Has a genome comprising at least one Jλ in at least one hVλ and λ light chain locus, and the λ light chain locus contains a non-human Cλ gene); (b) developing an immune response to the antigen in the genetically modified animal; And, (c) isolating an antibody that specifically recognizes the antigen (b) from an animal, or (b) isolating a cell comprising an immunoglobulin domain that specifically recognizes the antigen from an animal, or Identifying a B nucleic acid sequence encoding a heavy and/or light chain variable domain that binds an antigen, wherein the antibody comprises a light chain derived from hVλ, hJλ and non-human Cλ genes. In one embodiment, the non-human animal is selected from mice, rats, hamsters, sheep, goats, cattle and non-human primates.

In one embodiment, the λ light chain constant gene is selected from a human Cλ gene and a non-human Cλ gene. In one embodiment, the λ light chain constant gene is a human Cλ gene. In a specific embodiment, the human Cλ gene is selected from Cλ1, Cλ2, Cλ3 and Cλ7. In one embodiment, the λ light chain constant gene is a mouse or rat Cλ gene. In a specific embodiment, the mouse Cλ gene is selected from Cλ1, Cλ2 and Cλ3. In another specific embodiment, the mouse Cλ gene is Cλ2. In another specific embodiment, the mouse Cλ gene is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

In one aspect, (a) exposing the genetically modified non-human animal to an antigen, wherein the genetic modification includes hVλ and hJλ at the endogenous light chain site, and the endogenous light chain site is the non-human C _L gene or its functionality Including fragments); And (b) identifying the rearranged immunoglobulin gene in the non-human animal. Provided is a method for producing a rearranged antibody gene in a genetically modified non-human animal, wherein the rearranged immunoglobulin The gene includes a λ light chain variable region gene segment and a C _L gene or a functional fragment thereof.

In one embodiment, the method further comprises cloning a nucleic acid sequence encoding a heavy and/or light chain variable region from an animal, wherein the heavy and/or light chain variable region comprises human Vλ and mouse C _L. Derived from antibodies.

In one embodiment, the mouse C _L gene or a functional fragment thereof is selected from a human C _L gene and a mouse C _L gene, or a functional fragment thereof.

In one embodiment, a method of producing an antibody gene rearranged in a genetically modified non-human animal is provided, the method comprising: (a) exposing the genetically modified non-human animal to an antigen, wherein Contains hVλ and hJλ at the κ light chain site, and the κ light chain site includes a non-human Cκ gene or a functional fragment thereof); And (b) identifying the rearranged immunoglobulin gene in the animal, wherein the rearranged immunoglobulin gene includes a λ light chain variable region gene segment and a Cκ gene or a functional fragment thereof.

In one embodiment, the κ light chain constant gene or functional fragment thereof is selected from a human Cκ gene and a non-human (eg, mouse or rat) Cκ gene, or a functional fragment thereof.

In one embodiment, the method further comprises cloning a nucleic acid sequence encoding a heavy and/or light chain variable region from an animal, wherein the heavy and/or light chain variable region is human Vλ and non-human (e.g., Mouse or rat) is derived from an antibody comprising Cκ.

In one embodiment, a method for producing a rearranged antibody gene in a genetically modified non-human animal is provided, the method comprising: (a) exposing the genetically modified non-human animal to an antigen (wherein the gene The modification includes hVλ and hJλ in the non-human λ light chain site, and the λ light chain site includes the non-human Cλ gene or functional fragment thereof); And (b) identifying the rearranged immunoglobulin gene in the animal, wherein the rearranged immunoglobulin gene includes a λ light chain variable region gene segment and a Cλ gene or a functional fragment thereof.

In one embodiment, the λ light chain constant gene or a functional fragment thereof is selected from a human Cλ gene and a mouse or rat Cλ gene, or a functional fragment thereof. In a specific embodiment, the λ light chain constant gene is a mouse or rat Cλ gene, or a functional fragment thereof.

In one embodiment, the method further comprises cloning a nucleic acid sequence encoding a heavy and/or light chain variable region from an animal, wherein the heavy and/or light chain variable region is human Vλ and non-human (e.g., Mouse or rat) Cλ.

In one aspect, a method of producing an antibody is provided, wherein the method comprises exposing a non-human animal as described herein to an antigen, an immune response comprising producing an antibody that specifically binds to the antigen. In the animal, identifying the rearranged nucleic acid sequence in the animal encoding the heavy chain and the rearranged nucleic acid sequence in the animal encoding the cognate light chain variable domain sequence of the antibody (wherein the antibody specifically binds to an antigen) ), and using the nucleic acid sequences of heavy and light chain variable domains fused to a human constant domain, comprising the step of preparing an antibody of interest, wherein the antibody of interest comprises a Vλ domain fused to a _{C L domain.} It contains the light chain. In one embodiment, the Vλ domain is human and the C _L domain is a human or mouse or rat Cλ domain. In one embodiment, the Vλ domain is a mouse or rat and the C _L domain is a human or mouse CK domain.

In one embodiment, a method of producing an antibody is provided, the method comprising exposing a non-human animal as described herein to an antigen, and producing an antibody that specifically binds to the antigen. Developing a reaction in an animal, identifying the rearranged nucleic acid sequence in the animal encoding the heavy chain and the rearranged nucleic acid sequence in the animal encoding the cognate light chain variable domain sequence of the antibody, wherein the antibody is specifically antigen-specific. Conjugated), and using the nucleic acid sequence of the heavy and light chain variable domains fused to the nucleic acid sequence of the human constant domain, wherein the antibody of interest is a Vλ domain fused to the Cκ domain It includes a light chain containing.

In one embodiment, a method of producing an antibody is provided, the method comprising exposing a non-human animal as described herein to an antigen, and producing an antibody that specifically binds to the antigen. Developing a reaction in an animal, identifying a rearranged nucleic acid sequence encoding a heavy chain variable domain in an animal and a rearranged nucleic acid sequence encoding a cognate light chain variable domain sequence of the antibody, wherein the antibody is specifically antigen-specific. Conjugated), and using a nucleic acid sequence fused to a nucleic acid sequence encoding a human constant domain and a human light chain constant domain, wherein the antibody specifically bound to the antigen is It includes a light chain comprising a human Vλ domain fused to a non-human (eg, mouse or rat) Cλ region.

In one embodiment, the Cλ region is a mouse, and in one embodiment is selected from Cλ1, Cλ2 and Cλ3. In a specific embodiment, the mouse Cλ region is Cλ2.

In one aspect, a method of constructing a rearranged antibody light chain variable region gene sequence is provided, the method comprising: (a) exposing a non-human animal as described herein to an antigen; (b) developing an immune response in the animal; (c) identifying a cell in the animal comprising a nucleic acid sequence encoding a rearranged human Vλ domain sequence fused to a non-human C _{L domain, wherein the cell is also a human V H} domain and a non-human C _H domain. It encodes a cognate heavy chain comprising, and the cell expresses an antibody that binds to an antigen); (d) cloning from a nucleic acid sequence encoding a human Vλ domain and a nucleic acid sequence encoding a cognate human V _{H domain;} And, (e) constructing a complete human antibody using the cloned nucleic acid sequence encoding the human Vλ domain and the cloned nucleic acid sequence encoding the cognate human V _{H domain.} In one embodiment, the non-human animal and non-human domain are selected from mice and rats.

In one embodiment, a method of constructing a rearranged antibody light chain variable region gene sequence is provided, the method comprising: (a) exposing a non-human animal as described in the present disclosure to an antigen; (b) developing an immune response in the animal; (c) identifying a cell in an animal comprising a nucleic acid sequence encoding a human Vλ domain sequence sequentially rearranged on the same nucleic acid molecule carrying a nucleic acid sequence encoding a Cκ domain of a non-human animal, wherein the cell is _{It also encodes a cognate heavy chain comprising the human V H} domain and C _H domain of a non-human animal, and the cell expresses the antibody bound to the antigen); (d) cloning a sequence encoding a human Vλ domain and a nucleic acid sequence encoding a _{cognate human V H domain from the cellular nucleic acid;} And (e) constructing a fully human antibody using the cloned nucleic acid sequence encoding the human Vλ domain and the cloned nucleic acid sequence encoding the cognate human V _{H domain.}

In one embodiment, a method of constructing a rearranged antibody light chain variable region gene sequence is provided, the method comprising: (a) exposing a non-human animal as described herein to an antigen; (b) developing an immune response to the antigen in the animal; (c) identifying a cell in the animal comprising DNA encoding the rearranged human Vλ domain sequence fused to the animal's non-human Cλ domain, wherein the cell is also the animal's human V _H domain and the non-human C _{Encodes a cognate heavy chain comprising an H} domain and the cell expresses an antibody that binds to the antigen); (d) cloning a sequence encoding a human Vλ domain rearranged from a cellular nucleic acid and a nucleic acid sequence encoding a _{cognate human V H domain;} And (e) constructing a fully human antibody using the cloned nucleic acid sequence encoding the human Vλ domain and the cloned nucleic acid sequence encoding the cognate human V _{H domain.} In one embodiment, the non-human animal is a mouse and the Cλ domain is a mouse Cλ2. In a specific embodiment, the mouse Cλ domain is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

In one aspect, a genetically modified non-human animal expressing a human λ-derived light chain fused to an endogenous light chain constant region (C _L ) is provided, wherein the animal is, upon immunization with an antigen, the non-human of the animal An antibody comprising a human Vλ domain fused to a C _{L domain is constructed.} In one embodiment, the non-human C _L domain is selected from a Cκ domain and a Cλ domain. In one embodiment, the C _L domain is a Cκ domain. In one embodiment, the animal is a mouse. In one embodiment, the mouse C _L domain is a Cλ domain. In a specific embodiment, the Cλ domain is Cλ2. In a specific embodiment, the mouse Cλ domain is derived from a Cλ gene that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to mouse Cλ2.

In one aspect, a genetically modified non-human animal comprising a modified endogenous κ or λ light chain site as described herein is provided that expresses a plurality of immunoglobulin λ light chains associated with a plurality of immunoglobulin heavy chains. In one embodiment, the heavy chain comprises a human sequence. In various embodiments, the human sequence is selected from variable sequences, C _H 1, hinges, C _H 2, C _H 3, and combinations thereof. In one embodiment, the plurality of immunoglobulin λ light chains comprises human sequences. In various embodiments, the human sequence is selected from variable sequences, constant sequences, and combinations thereof. In one embodiment, the animal comprises an incompetent endogenous immunoglobulin site and expresses a heavy chain and/or a λ light chain from a transgene or extrachromosomal episome. In one embodiment, the animal is a minor or all endogenous non-human heavy chain gene segment (i.e., V, D, J) and/or some or all of an endogenous non-human heavy chain constant sequence (e.g. , C _H 1, hinge, C _H 2, C _H 3, or combinations thereof), and/or some or all of And replacement of an endogenous non-human light chain sequence (eg , V, J, constant, or combinations thereof) with one or more human immunoglobulin sequences. In one embodiment, the non-human animal is a mouse.

In one aspect, a non-human animal suitable for constructing an antibody with a human lambda-derived light chain is provided, wherein all or substantially all of the antibodies produced in the non-human animal are expressed as a human lambda-derived light chain. In one embodiment, the human lambda-derived light chain is expressed from an endogenous light chain locus. In one embodiment, the endogenous light chain site is a κ light chain site. In a specific embodiment, the animal is a mouse and the κ light chain site is a mouse κ light chain site.

In one aspect, a method for constructing a λ-derived light chain for a human antibody is provided, the method comprising obtaining a light chain sequence and a heavy chain sequence from a non-human animal as described herein, and preparing a human antibody. In this step, the light chain sequence and the heavy chain sequence are used.

In one aspect, a method of constructing an antigen-binding protein is provided, the method comprising: exposing a non-human animal as described herein to an antigen; Developing an immune response in a non-human animal; And obtaining an antigen-binding protein bound to an antigen from a non-human animal, or obtaining a sequence to be used in preparing an antigen-binding protein that binds the antigen from a non-human animal.

In one aspect, cells derived from a non-human animal (eg, mouse or rat) as described herein are provided. In one embodiment, the cells are selected from embryonic stem cells, pluripotent cells, induced pluripotent cells, B cells and hybridomas.

In one aspect, a cell comprising a genetic modification as described herein is provided. In one embodiment, the cell is a mouse cell. In one embodiment, the cell is selected from hybridoma and quadroma. In one embodiment, the cell expresses an immunoglobulin light chain comprising a human λ variable sequence fused to a mouse constant sequence. In a specific embodiment, the mouse constant sequence is a mouse κ constant sequence.

In one aspect, tissue derived from a non-human animal as described herein is provided.

In one aspect, there is provided the use of a non-human animal or cell as described herein for making an antigen-binding protein. In one embodiment, the antigen-binding protein is a human protein. In one embodiment, the human protein is a human antibody.

In one aspect, an antigen-binding protein produced by a non-human animal, cell, tissue or method as described herein is provided. In one embodiment, the antigen-binding protein is a human protein. In one embodiment, the human protein is a human antibody.

Any of the embodiments and aspects described herein may be used in connection with each other, unless otherwise stated or apparent from the context. Other embodiments will become apparent to those skilled in the art from review of the description that follows.

1A is a constant scale of direct genomic replacement of about 3 megabases (Mb) of a mouse immunoglobulin heavy chain variable locus (black symbol) to about 1 megabase (Mb) of a human immunoglobulin heavy chain variable locus (white symbol). This is not a rough diagram.
1B is the first of about 0.5 megabases (Mb) of two approximately identical repeats of the human immunoglobulin κ light chain variable locus of the mouse immunoglobulin κ light chain variable locus (black symbol) of about 3 megabases (Mb). Or it represents a schematic, not to scale, direct genome replacement to the proximal (white symbol).
Figure 2a is a direct genomic replacement of a mouse immunoglobulin heavy chain variable locus resulting in deletion of all mouse V _H , D _H and J _H gene segments and replacement with three human V _H , all human D _H and J _{H gene segments.} A schematic, not to scale, schematic diagram of the three initial stages (A to C) of is shown. The targeting vector for the λ insert of the human immunoglobulin heavy chain gene segment was a 67 kb 5'mouse homology segment, a selection cassette (white square), a site-specific recombination site (white triangle), a 145 kb human genome fragment and an 8 kb. Shown together with the 3'mouse homology section (3hV _H BACvec). Human (white symbols) and mouse (black symbols) immunoglobulin gene segments inserted from subsequent targeting vectors, additional selection cassettes (white squares) and site-specific recombination sites (white triangles) are shown.
Figure 2B is a constant scale of six additional steps (D to I) for direct genomic replacement of the mouse immunoglobulin heavy chain variable locus resulting in the insertion of 77 additional human V _{H gene segments and removal of the last selection cassette.} Rather, it shows a detailed illustration. The targeting vector for the insert of the additional human V _{H gene segment (18hV H} BACvec) to the early insert of the human heavy chain gene segment (3hV _H -CRE hybrid allele) is a 20 kb 5'mouse homology section, selection cassette (White square), overlapping the 5'end of the initial insert of the human heavy chain gene segment, shown as a 196 kb human genome fragment and a site-specific recombination site (white triangle) located 5'to the human gene segment. 62 kb human homology section. Human (white symbols) and mouse (black symbols) immunoglobulin gene segments and additional selection cassettes (white squares) inserted by subsequent targeting vectors are shown.
Figure 2c is a constant, three initial stages (A to C) of direct genomic replacement of the mouse immunoglobulin κ light chain variable locus resulting in deletion of all mouse Vκ, and Jκ gene segments (Igκ-CRE hybrid alleles). Shows detailed illustrations, not to scale. Selection cassettes (white squares) and site-specific recombination sites (white triangles) inserted from the targeting vector are shown.
Figure 2d shows the insertion of all human Vκ and Jκ gene segments in the proximal repeat and four additional for direct genomic replacement of the mouse immunoglobulin κ light chain variable locus resulting in deletion of the last selection cassette (40hVκdHyg hybrid allele). A detailed illustration of steps D to H is shown, not to scale. Human (white symbols) and mouse (black symbols) immunoglobulin gene segments and additional selection cassettes (white squares) inserted by subsequent targeting vectors are shown.
Figure 3a is a screening strategy comprising the location of a quantitative PCR (qPCR) primer/probe for direct insertion of human heavy chain gene sequence and loss of mouse heavy chain gene sequence in targeted embryonic stem (ES) cells, constant It is not a scale, but a schematic illustration. The strategy of selection of ES cells and mice for the first human heavy chain gene insertion is the unmodified mouse chromosome (top) and the deleted region (“lost” probe C and D), qPCR primers/probe sets for the insertion regions (“high H” probes G and H) and flanking regions (“retaining” probes A, B, E and F).
3B shows a representative calculation of the observed number of probe copies in parental and modified ES cells for a first insertion of a human immunoglobulin heavy chain gene segment. The observed probe copies for probes A to F were calculated as 2/2ΔΔCt. ΔΔCt is calculated as ave[ΔCt(sample)-medΔCt(control)], where ΔCt is the difference between the test probe and the reference probe (between 4 and 6 reference probes depending on the test). The term medΔCt (control) is the median ΔCt of multiple (>60) untargeted DNA samples from parental ES cells. Each modified ES cell clone was assayed in 6 pairs. To calculate the copy number of IgH probes G and H in parental ES cells, these probes were assumed to have a copy number of 1 and a maximum Ct of 35 in the modified ES cells even if no amplification was observed.
3C shows a representative calculation of the number of copies for 4 mice of each genotype calculated using only probes D and H. Wild-type mouse: WT mouse; Mouse heterozygous for the first insertion of a human immunoglobulin gene segment: HET mouse; Mouse homozygotes for the first insertion of a human immunoglobulin gene segment: Homo mice.
4A shows a detailed, not to scale, three-step, detailed illustration used in the construction of _{3hV H} BACvec by bacterial homologous recombination (BHR). Human (white symbols) and mouse (black symbols) immunoglobulin gene segments, selection cassettes (white squares) and site-specific recombination sites (white triangles) inserted from the targeting vector are shown.
4B shows pulse-field gel electrophoresis (PFGE) of three BAC clones (B1, B2 and B3) after NotI digestion. Markers M1, M2 and M3 are the low range, mid range and lambda ladder PEG markers, respectively (New England BioLabs, Ipswich, Mass.).
Figure 5a shows A schematic, not to scale, schematic illustration of a sequential modification of a mouse immunoglobulin heavy chain locus while increasing the amount of human immunoglobulin heavy chain gene segments. Homozygous mice were constructed from each of the three different stages of heavy chain humanization. White symbols represent human sequences; Black symbols indicate mouse sequences.
Figure 5b is A schematic, not to scale, schematic illustration of the sequential modification of the mouse immunoglobulin κ light chain locus while increasing the amount of human immunoglobulin κ light chain gene segments. Homozygous mice were constructed from each of the three different stages of κ light chain humanization. White symbols represent human sequences; Black symbols indicate mouse sequences.
6 shows FACS dot plots of B cell populations in wild-type and VELOCIMMUNE® humanized mice. Spleen (first row, third and last row from the top) or wild type (wt), VELOCIMMUNE® 1(V1), VELOCIMMUNE® 2(V2) or velocimune ( VELOCIMMUNE) (registered trademark) 3(V3) Cells from the inguinal lymph nodes (second row from top) of mice are surface IgM-expressing B cells (first row and second row from top), surface immunoglobulins containing κ or λ light chains. (Third line from top) or specific monophasic surface IgM (last line), and stained for the population separated by FACS.
7A shows a representative heavy chain CDR3 sequence of a randomly selected VELOCIMMUNE® antibody around the _{V H} -D _H -J _{H (CDR3) junction, demonstrating the conjugation diversity and nucleotide addition.} The heavy chain CDR3 sequences are _{grouped according to the use of the D H} gene segment, the germline of which is presented in bold above each group. _{The V H} gene segment for each heavy chain CDR3 sequence is indicated in parentheses at the 5'end of each sequence (eg, 3-72 is human V _H 3-72). _{The J H} gene segment for each heavy chain CDR3 is indicated in parentheses at the 3'end of each sequence (eg, 3 is human J _H 3). The SEQ ID NOs for each sequence shown are as follows from top to bottom: SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39.
7B shows a representative light chain CDR3 sequence of a randomly selected VELOCIMMUNE® antibody around the Vκ-Jκ(CDR3) junction, demonstrating junction diversity and nucleotide addition. The Vκ gene segment for each light chain CDR3 sequence is indicated in parentheses at the 5'end of each sequence (eg, 1-6 is human Vκ1-6). The Jκ gene segment for each light chain CDR3 sequence is indicated in parentheses at the 3'end of each sequence (eg, 1 is human Jκ1). SEQ ID NOs for each sequence shown are as follows from top to bottom: SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58.
Figure 8 shows each of a set of 38 (non-immunized IgM), 28 (non-immunized IgG), 32 (non-immunized Igκ from IgG), 36 (immunized IgG) or 36 (non-immunized Igκ from IgG) sequence. Somatic cells of heavy and light chains of VELOCIMMUNE (registered trademark) antibodies scored as percent of the sequence changed at the nucleotide (NT; left column) or amino acid (AA; right column) position (after alignment to the mating germline sequence). It represents the somatic hypermutation frequency. Shaded bars indicate the location of the CDRs.
9A shows the levels of serum immunoglobulins against IgM and IgG isotypes in wild-type (white bars) or VELOCIMMUNE® mice (black bars).
9B shows the levels of serum immunoglobulins against the IgA isotype of green type (white bars) or VELOCIMMUNE® mice (black bars).
9C shows the levels of serum immunoglobulins against the IgE isotype of wild-type (white bars) or VELOCIMMUNE® mice (black bars).
FIG. 10A is from 7 VELOCIMMUNE (registered trademark) (VI) and 5 wild-type (WT) mice after 2 (cross 1) or 3 times (cross 2) immunizations with the ectodomain of IL-6R. Antigen-specific IgG titers to the interleukin-6 receptor (IL-6R) in the serum of are shown.
10B shows IL-6R-specific IgG isotype-specific titers from 7 VELOCIMMUNE® (VI) and 5 wild type (WT) mice.
Fig. 11A shows the affinity distribution of anti-interleukin-6 receptor monoclonal antibodies generated in VELOCIMMUNE® mice.
11B shows antigen-specific blocking of anti-interleukin-6 receptor monoclonal antibodies generated in VELOCIMMUNE® (VI) and wild-type (WT) mice.
Figure 12 shows a schematic, not to scale, a constant scale of the mouse ADAM6a and ADAM6b genes at the mouse immunoglobulin heavy chain site. Targeting vectors (mADAM6 targeting vectors) used for insertion of mouse ADAM6a and ADAM6b into humanized endogenous heavy chain sites contain site-specific recombination sites (Frt) comprising engineered restriction sites on the 5'and 3'ends. Shown as a selection cassette (HYG: hygromycin) on the side.
Figure 13 is a schematic, not to scale, human ADAM6 pseudogene (hADAM6Y) located between human heavy chain variable gene segments 1-2 (V _H 1-2) and 6-1 (V _{H 6-1).} Show. The targeting vector for bacterial homologous recombination (hADAM6Y targeting vector) to delete the human ADAM6 pseudogene and insert a specific restriction site into the human heavy chain site is a site containing engineered sites on the 5'and 3'ends- It is shown as a selection cassette (NEO: neomycin) with a specific recombination site (loxP) on both sides. A non-scale schematic of the resulting targeted humanized heavy chain sites containing genomic fragments encoding the mouse ADAM6a and ADAM6b genes comprising a selection cassette with site-specific recombination sites on both sides is shown.
Figure 14a is a mouse homozygous for human heavy chain and human κ light chain variable locus (H ^{+ / +} κ ^{+ / +} ) and an inserted mouse genome comprising a mouse ADAM6 gene (H ^{+ / +} A6 ^res κ ^{+ / +)} FACS contour plots of lymphocytes gated on a single line for the surface expression of IgM and B220 in the bone marrow for mouse homozygotes for human heavy chain and human κ light chain variable loci with fragments are shown. The percentages of immature (B220 ^int IgM ⁺ ) and mature (B220 ^high IgM ⁺ ) B cells are indicated on each contour plot.
Figure 14B is a human heavy chain and human κ light chain variable locus ^{carrying an ectopic mouse genomic fragment encoding a mouse homozygous (H + / +} κ ^{+ / +} ) and mouse ADAM6 gene for the human heavy and human κ light chain variable locus. Shows the total number ^{of immature (B220 int} IgM ⁺ ) and mature (B220 ^high IgM ⁺ ) B cells in the bone marrow isolated from the femur of the mouse homozygote (H ^+/+ A6 ^res κ ^{+/+) for the mouse.}
Figure 15A is a human heavy chain and a human κ light chain variable locus ^{carrying an ectopic mouse genomic fragment encoding a mouse homozygous (H + / +} κ ^{+ / +} ) and a mouse ADAM6 gene for the human heavy chain and human κ light chain variable locus. ^{FACS contour plots of CD19 +} -gated B cells for the surface expression of c-kit and CD43 in the bone marrow of the mouse homozygous for (H ^+/+ A6 ^res κ ^+/+ ) are shown. Pro -B (CD19 ⁺ CD43 ⁺ kit ⁺ c) and presence -B (CD19 ^{+ CD43-kit c-)} percentage of cells is indicated on each of the upper right and lower left quadrants of each contour diagram.
Figure 15b is a human heavy chain and human κ light chain variable locus ^{having an ectopic mouse genomic fragment containing a mouse homozygous (H + / +} κ ^{+ / +} ) and mouse ADAM6 gene for human heavy chain and human κ light chain variable locus mouse homozygous for the (H ^{+ / +} A6 ^res κ ^{+ / +)} cells in the bone marrow pro -B isolated from the femur (CD19 ⁺ CD43 ⁺ kit ⁺ c) and presence -B cells (CD19 ^{+ CD43-kit c-} Represents the total number of ).
Figure 16A is a human heavy chain and human κ light chain variable locus with mouse homozygous for human heavy and human κ light chain variable loci (H ^+/+ κ ^+/+ ) and ectopic mouse genomic fragments encoding the mouse ADAM6 gene. FACS contour plots of lymphocytes gated on a single line are shown for the surface expression of CD19 and CD43 in the bone marrow of the mouse homozygote (H ^+/+ A6 ^res κ ^{+/+) for.} Immature B (CD19 ⁺ CD43 ^-) Percentage of pre -B (CD19 ⁺ CD43 ^int) and Pro -B (CD19 ⁺ CD43 ⁺⁾ is indicated on each of the contour diagram.
Figure 16B is a human heavy chain and human κ light chain variable locus with mouse homozygous (H ^+/+ κ ^+/+ ) for human heavy and human κ light chain variable loci and an ectopic mouse genomic fragment encoding the mouse ADAM6 gene. for mice homozygous ^{(H + / + A6 res κ} + / +) bone marrow immature B (CD19 ⁺ CD43 ^-) in the presence of, and -B (CD19 ⁺ CD43 ^int) denotes a histogram in the cell.
Figure 17A is a human heavy chain and human κ light chain variable locus with mouse homozygotes (H ^+/+ κ ^+/+ ) for human heavy and human κ light chain variable loci and ectopic mouse genomic fragments encoding the mouse ADAM6 gene. FACS contour plots of lymphocytes gated on a single line for the surface expression of CD19 and CD3 in splenocytes for mouse homozygous for (H ^+/+ A6 ^res κ ^{+/+) are shown.} B (CD19 ⁺ CD3 ^-) and T (CD19 ^- CD3 ⁺⁾ percentage of cells is indicated on each of the contour diagram.
Figure 17B is a human heavy chain and human κ light chain variable locus with mouse homozygous for human heavy and human κ light chain variable loci (H ^+/+ κ ^+/+ ) and ectopic mouse genomic fragments encoding the mouse ADAM6 gene. ^{Contour plots of FACs for CD19 +} -gated B cells are shown for the surface expression of Igλ and Igκ light chains in the spleen of the mouse homozygote (H ^+/+ A6 ^res κ ^{+/+) against.} The percentages of Igλ ⁺ (upper left quadrant) and Igκ ⁺ (lower right quadrant) B cells are plotted on each contour plot.
Figure 17C is a human heavy chain and human κ light chain variable locus with mouse homozygous for human heavy and human κ light chain variable loci (H ^+/+ κ ^+/+ ) and ectopic mouse genomic fragments encoding the mouse ADAM6 gene. It shows the total number ^{of CD19 +} B cells in the spleen of the mouse homozygote (H ^{+ / +} A6 ^res κ ^{+ / +) for.}
Figure 18A is a human heavy chain and human κ light chain variable locus ^{carrying an ectopic mouse genomic fragment encoding a mouse homozygous (H + / +} κ ^{+ / +} ) and mouse ADAM6 gene for the human heavy and human κ light chain variable locus. Contour plots of FACs of ^{CD19 +} -gated B cells are shown for the surface expression of IgD and IgM in the spleen of the mouse homozygote (H ^+/+ A6 ^res κ ^{+/+) against.} The percentage of mature B cells (CD19 ⁺ IgD ^high IgM ^int ) is indicated on each contour plot. Arrows on the right contour diagram illustrate the maturation process of B cells related to IgM and IgD surface expression.
Figure 18b is a mouse homozygous for the human heavy chain and human κ light chain variable locus during maturation ^{from CD19 +} IgM ^high IgD ^int to CD19 ⁺ IgM ^int IgD ^{high (H + / +} κ ^{+ / +} ) and mouse ADAM6 gene encoding. The total number of B cells in the spleen of ^{mouse homozygous (H +/+} A6 ^res κ ^+/+ ) carrying ectopic mouse genomic fragments is shown.
Fig. 19 shows a detailed, not to scale, a detailed illustration of the human λ light chain locus, including the Vλ gene segments (A, B and C) and closters of the Jλ and Cλ region pairs (JC pairs).
Figure 20 shows a schematic, not to scale, a targeting strategy used to inactivate the endogenous mouse λ light chain site.
Figure 21 shows a schematic, not to scale, a targeting strategy used to inactivate the endogenous mouse κ light chain site.
Figure 22a is a rough illustration, not to scale, of an initial targeting vector for targeting an endogenous mouse λ light chain site with a human λ light chain sequence comprising a 12 hVλ gene segment and an hJλ1 gene segment (12/1-λ targeting vector). Represents.
Figure 22b is a 12 hVλ gene segment and hJλ1 gene segment (12/1-κ targeting vector), 12 hVλ gene segment and hJλ1, 2, 3 and 7 gene segments (12/4-κ targeting vector), 12 hVλ gene segment, Human Vκ-Jκ genomic sequence and hJλ1 gene segment (12(κ)1-κ targeting vector) and 12 hVλ gene segment, human Vκ-Jκ genomic sequence and hJλ1, 2, 3, and 7 gene segment (12(κ)4- κ targeting vectors), showing a schematic, not to scale, four initial targeting vectors targeting endogenous mouse κ light chain sites with human λ light chain sequences.
23A shows a schematic, not to scale, a targeting strategy for progressively inserting 40 hVλ gene segments and a single hJλ gene segment into the mouse λ light chain site.
23B shows a schematic, not to scale, a targeting strategy for progressively inserting 40 hVλ gene segments and a single hJλ gene segment into a mouse κ site.
Figure 24 shows a constant scale of targeting and molecular engineering steps used to construct a unique human λ-κ hybrid targeting vector for the construction of a hybrid light chain site containing human κ intergenic sequences, multiple hJλ gene segments, or both. Rather, it represents a rough illustration.
25A shows a schematic, not to scale, locus structure for a modified mouse lambda light chain locus containing 40 hVλ gene segments and a single hJλ gene segment operably linked to the endogenous Cλ2 gene.
Figure 25B shows 4 independent modified mouse κ light chain sites containing 1 to 4 Jλ gene segments and 40 hVλ gene segments with or without a continuous human Vκ-Jκ genomic sequence operably linked to an endogenous Cκ gene. It represents a rough illustration, not to scale, of the site structure.
Figure 26a is a wild-type mouse (WT), mouse homozygous for 12 hVλ and 4 hJλ gene segments containing human Vκ-Jκ genome sequences (12hVλ-VκJκ-4hJλ) and 40 hVλ and 1 hJλ gene segments. ^{Contour plots of Igλ +} and Igκ ⁺ splenocytes gated for ^{CD19 +} from mouse homozygotes (40hVλ-1hJλ) for.
26B is a wild-type (WT), mouse homozygous (12hVλ-VκJκ-4hJλ) for 12 hVλ and 4 hJλ gene segments comprising the human Vκ-Jκ genomic sequence and 40 hVλ and 1 hJλ gene segments. The total number ^{of CD19 +} B cells in the harvested spleen from mouse homozygous (40hVλ-1hJλ) is shown.
Figure 27A shows, in the top panel, B and T cells from wild-type mouse (WT) and mouse homozygotes (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence. Contour plots of gated and stained splenocytes on a single line for (CD19 ⁺ and CD3 ^{+, respectively) are shown.} The lower panel shows CD19 for ^{Igλ +} and ^{Igκ +} expression from wild-type mouse (WT) and mouse homozygotes (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence. Contour plots of splenocytes gated and stained on ^{+ are shown.}
Figure 27B ^{shows CD19 +} , CD19 ⁺ in spleens harvested from mouse homozygotes (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments containing wild-type mouse (WT) and human Vκ-Jκ genomic sequences. Shows the total number of ^Igκ+ and CD19 ^{+Igλ+ B cells.}
Figure 27C shows immunoglobulin D (IgD) and immunoglobulins from mouse homozygotes (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments comprising wild-type mouse (WT) and human Vκ-Jκ genomic sequences. Contour plots of splenocytes gated and stained on ^{CD19 +} for M(IgM) are shown. Mature (72 for WT, 51 for 40hVλ-VκJκ-4hJλ) and transient (13 for WT, 22 for 40hVλ-VκJκ-4hJλ) B cells are indicated on each of the contour plots.
Figure 27D ^{is a wild-type mouse (WT) and CD19 +} B cells in the spleen harvested from the mouse homozygous (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments containing the human Vκ-Jκ genomic sequence, transient. The total number of red B cells (CD19 ⁺ IgM ^hi IgD ^lo ) and mature B cells (CD19 ⁺ IgM ^lo IgD ^hi ) is shown.
28A shows, in the top panel, B and T cells from wild-type mouse (WT) and mouse conjugates (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments comprising the human Vκ-Jκ genomic sequence ( Contour plots of the stained bone marrow are shown for CD19 ⁺ and CD3 ^{+, respectively.} The lower panel is gated on ^{CD19 +} from mouse homozygotes (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments comprising wild-type mouse (WT) and human Vκ-Jκ genomic sequences and ckit ⁺ And a contour plot of the bone marrow stained for ^{CD43 +.} Pro and pre B cells are plotted against the contour plot in the lower panel.
Figure 28b is a wild-type mouse (WT) and a mouse homozygous for 40 hVλ and 4 Jλ gene segments containing the human Vκ-Jκ genomic sequence (40hVλ-VκJκ-4hJλ) pro (CD19 ^{+) in bone marrow harvested from the femur.} CD43 ⁺ kit ⁺ c) and presence (CD19 ^{+ CD43-C} kit ^-) indicates the number of B cells.
Figure 28c is for immunoglobulin M (IgM) and B220 of mouse homozygous (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments comprising wild-type mouse (WT) and human Vκ-Jκ genomic sequences. A contour plot of the bone marrow gated on a stained single line is shown. Immature, mature and pro/pre B cells are marked on each of the contour diagrams.
Figure 28d ^{shows the immature (B220 int} ) in the bone marrow isolated from the femur of the mouse homozygous (40hVλ-VκJκ-4hJλ) for 40 hVλ and 4 Jλ gene segments containing wild-type mouse (WT) and human Vκ-Jκ genomic sequences. Shows the total number of IgM ⁺ ) and mature (B220 ^hi IgM ^{+) B cells.}
Figure 28E is a wild-type mouse (WT) and human Vκ-Jκ for 40 hVλ and 4 Jλ gene segments containing the genomic sequence of mouse homozygotes (40hVλ-VκJκ-4hJλ) for Igλ and Igκ expression isolated from the femur Contour plots of bone marrow gated for stained immature (B220 ^int IgM ⁺ ) and mature (B220 ^hi IgM ^{+) B cells are shown.}
29 shows the nucleotide sequence alignment of the Vλ-Jλ-Cκ junction of eight independent RT-PCR clones amplified from mouse splenocyte RNA carrying the human λ light chain gene sequence at the endogenous mouse κ light chain locus. A6 = SEQ ID NO: 115; B6 = SEQ ID NO: 116; F6 = SEQ ID NO: 117; B7 = SEQ ID NO: 118; E7 = SEQ ID NO: 119; F7 = SEQ ID NO: 120; C8 = SEQ ID NO: 121; E12 = SEQ ID NO: 122; 1-4 = SEQ ID NO: 123; 1-20 = SEQ ID NO: 124; 3B43 = SEQ ID NO: 125; 5-8 = SEQ ID NO: 126; 5-19 = SEQ ID NO: 127; 1010 = SEQ ID NO: 128; 11A1 = SEQ ID NO: 129; 7A8 = SEQ ID NO: 130; 3A3 = SEQ ID NO: 131; 2-7 = SEQ ID NO: 132. Lowercase bases represent non-germline bases resulting from mutations and/or N additions during recombination. The consensus amino acid in the framework 4 region (FWR4) encoded by the nucleotide sequence of hJλ1 and mouse Cκ is indicated at the bottom of the sequence alignment.
Figure 30 is the Vλ-Jλ-Cκ junction of 12 independent RT-PCR clones amplified from splenocyte RNA of a mouse carrying a human λ light chain gene sequence comprising a contiguous human Vκ-Jκ genomic sequence at the endogenous mouse κ light chain locus. The nucleotide sequence alignment of is shown. 5-2 = SEQ ID NO: 145; 2-5 = SEQ ID NO: 146; 1-3 = SEQ ID NO: 147; 4B-1 = SEQ ID NO: 148; 3B-5 = SEQ ID NO: 149; 7A-1 = SEQ ID NO: 150; 5-1 = SEQ ID NO: 151; 4A-1 = SEQ ID NO: 152; 11A-1 = SEQ ID NO: 153; 5-7 = SEQ ID NO: 154; 5-4 = SEQ ID NO: 155; 2-3 = SEQ ID NO: 156. Lowercase bases represent non-germline bases resulting from mutations and/or N additions during recombination. The consensus amino acids in the framework 4 region (FWR4) encoded by the nucleotide sequence of each human Jλ and mouse Cκ are indicated at the bottom of the sequence alignment.
Figure 31 shows the nucleotide sequence alignment of the Vλ-Jλ-Cλ junction of three independent RT-PCR clones amplified from mouse splenocyte RNA carrying the human λ light chain gene sequence at the endogenous mouse λ light chain locus. 2D1 = SEQ ID NO: 159; 2D9 = SEQ ID NO: 160; 3E15 = SEQ ID NO: 161. Lowercase bases represent non-germline bases resulting from mutations and/or N additions during recombination. A consensus amino acid in the framework 4 region (FWR4) encoded by the nucleotide sequence of hJλ1 and mouse Cλ2 is indicated below the sequence alignment.

The present invention is not limited to the specific methods and experimental conditions described, and these methods and conditions may vary. It should be understood that the terms described herein are for the purpose of describing particular embodiments only, and are not intended to be limiting, as the scope of the invention is defined by the claims.

Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have achieved in the art, unless clearly indicated to the contrary or if the term or phrase is not clearly apparent from the context in which it is used. Any methods and materials similar or identical to those described herein can be used in the practice or testing of the present invention, but specific methods and materials are now described. All mentioned publications are incorporated herein by reference.

The phrase “substantially” or “substantially” when used to refer to an amount of a gene segment (eg, “substantially all” V gene segments) includes both functional and non-functional gene segments, and in various embodiments , For example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all gene segments; In various embodiments, “substantially all” gene segments, for example, contain at least 95%, 96%, 97%, 98% or 99% functional (ie, non-pseudogene) gene segments. Includes.

The term “replacement” includes a DNA sequence being located within the genome of a cell to replace a sequence within the genome with a heterologous sequence (eg, a human sequence in a mouse) at the site of the genomic sequence. The DNA sequence so positioned may include one or more regulatory sequences (e.g., promoters, enhancers, 5'or 3'untranslated regions, appropriate recombination signal sequences, etc.) that are portions of the source DNA used to obtain the positioned sequences. have. For example, in various embodiments, the replacement is a substitution of an endogenous sequence for a heterologous sequence that results in the production of a gene product from the DNA sequence so located (including a heterologous sequence), but not the expression of the endogenous sequence; The replacement has an endogenous genomic sequence with a DNA sequence encoding a protein with a similar function as a protein encoded by the endogenous genomic sequence (e.g., the endogenous genomic sequence encodes an immunoglobulin gene or domain, and the DNA fragment is one or more human Encoding an immunoglobulin gene or domain). In various embodiments, the endogenous gene or fragment thereof is replaced with a corresponding human gene or fragment thereof. The corresponding human gene or fragment thereof is an ortholog, homolog, or human gene or fragment of the replaced endogenous gene or fragment thereof, or the rescue and/or function is substantially the same as or identical to the replaced endogenous gene or fragment thereof.

The term “contiguous” (adjacent) includes reference to what occurs on the same nucleic acid molecule, eg , two nucleic acid sequences are contiguous unless they occur on the same nucleic acid molecule, but are not blocked by another nucleic acid sequence. For example, the rearranged V(D)J sequence has the constant region gene sequence, even if immediately after the final codon of the V(D)J sequence is not the first codon of the constant region sequence. In another example, two V gene segment sequences can be separated by a sequence that does not encode a codon in the V region, if they occur on the same genomic fragment, e.g., they are a promoter or other non-coding sequence. It is "continuous," even if it can be separated by regulatory sequences such as. In one embodiment, the contiguous sequence comprises genomic fragments containing genomic sequences that are arranged as found in the wild-type genome.

The phrase “derived from” when used for a variable region “derived” from a recited gene or gene segment, the original specific unrearranged gene segment or rearranged gene segment to form a gene expressing the variable domain Includes the ability to track sequences in (explain splice differences and somatic mutations, if applicable).

The phrase “functional” refers to use in an expressed antibody repertoire when used with respect to a variable region gene segment or a bound gene segment; For example, human Vλ gene segments 3-1, 4-3, 2-8, etc. are functional, whereas Vλ gene segments 3-2, 3-4, 2-5, etc. are non-functional.

“Heavy chain locus” refers to the location where the heavy chain variable (V _H ), heavy chain diversity (D _H ), heavy chain binding (J _H ) and heavy chain constant (C _H ) region DNA sequences in wild-type mice, on a chromosome, such as a mouse chromosome, are found. Includes.

The “κ site” includes the location where the κ variable (Vκ), κ binding (Jκ), and κ constant (Cκ) region DNA sequences are found in wild-type mice, on a chromosome, such as a mouse chromosome.

The “λ site” includes the site at which the λ variable (Vλ), λ binding (Jλ), and λ constant (Cλ) region DNA sequences are expressed in wild-type mice on a chromosome, such as a mouse chromosome.

The term “cell” when used in connection with expressing a sequence includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells (single or multicellular), bacterial cells (eg , E. coli , Bacillus spp. , Streptomyces spp. , etc.) , mycobacterial cells, fungi, etc. Cells, yeast cells (e.g. , S. cerevisiae , S. pombe ), Pichia pastoris , Pichia mentanolica ( P. methanolica ) Strains), plant cells, insect cells (e.g. , SF-9, SF-21, baculovirus -infected insect cells , Trichoplusia ni , etc.), non-human animal cells, human cells, B Cells, or cell fusions, such as cells of a hybridoma or quadroma. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In certain embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (eg , CHO K1, DXB-11 CHO, Veggie-CHO), COS (eg , COS-7), retina Cells, Vero, CV1, kidney (e.g. , HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g. , BHK21 ), Jurkat, Daudi, A431 (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, Tumor cells, and cell lines derived from the aforementioned cells. In certain embodiments, the cells comprise retinal cells (eg, PER.C6™ cells) that express one or more viral genes, such as viral genes.

The phrase “complementarity determining region” or the term “CDR” refers to an immunoglobulin gene of an organism (ie, a wild-type animal) that normally appears between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (eg, an antibody or T cell receptor). In) it includes an amino acid sequence encoded by a nucleic acid sequence. CDRs can be encoded, for example, by germline sequences or by rearranged or unrearranged sequences, and by, for example, naive or mature B cells or T cells. In some situations (e.g. , for CDR3), CDRs are not contiguous (e.g., in unrearranged nucleic acid sequences) as a result of splicing or linking sequences (e.g., VDJ recombination to form heavy chain CDR3), However, in a B cell nucleic acid sequence, it may be encoded by two or more consecutive sequences (eg, germline sequences).

The phrase “gene segment” or “segment” includes reference to a V (light or medium) or D or J (light or medium) immunoglobulin gene segment, which is a rearranged V/J or V/D/J sequence It includes sequences that have not been rearranged at immunoglobulin sites (eg, in humans and mice) capable of participating in rearrangements to form (eg, mediated by endogenous recombination). Unless otherwise indicated, the V, D and J segments contain a recombination signal sequence (RSS) that permits V/J recombination or V/D/J recombination according to the 12/23 rule. Unless otherwise indicated, segments further include sequences to which they are linked in nature or functional equivalents thereof (eg, for V segment promoter(s) and leader(s)).

The term “unrearranged” includes the state of the immunoglobulin site, wherein the V gene segment and the J gene segment (for heavy chain, D gene segment) remain separate, but a single V of the V(D)J repertoire, (D), can be combined to form a rearranged V(D)J gene, including J.

The phrase “micromolar range” is intended to mean 1 to 999 micromolar; The phrase “nanomolar range” is intended to mean 1 to 999 nanomolar; The phrase “picomolar range” is intended to mean 1 to 999 picomolar.

The term “non-human animals” is intended to include non-human animals such as protozoans, bony fish, cartilaginous fish such as sharks and rays, amphibians, reptiles, mammals and birds. Suitable non-human animals include mammals. Suitable mammals include non-human primates, goats, sheep, pigs, dogs, cattle and rodents. Suitable non-human animals are selected from the rodent family, including rats and mice. In one embodiment, the non-human animal is a mouse.

As a genetic model, mice have been greatly improved by transformation and knockout techniques, which allow to study the effects of directed overexpression or deletion of specific genes. Despite all its advantages, mice still have genetic disorders, which make them an incomplete model of human disease, making them an incomplete platform for testing and creating human therapeutics. First of all, about 99% of human genes have mouse homologs (Waterston, RH et al. (2002) Initial sequencing and comparative analysis of the mouse genome.Nature 420, 520-562.), but potential therapeutics are often intended for use. Failure to cross-react or inappropriately cross-react with mouse orthologs of human targets. To eliminate this problem, a selected target gene can be "humanized", ie, a mouse gene can be removed and replaced by the corresponding human ortholog gene sequence (e.g. , US Pat. 6,586,251, US 6,596,541 and US 7,105,348). Initially, efforts to humanize mouse genes by the "knockout-plus-transgenic humanization" strategy were the crossover of mice carrying deletions (ie knockouts) of endogenous genes with mice carrying randomly integrated human transgenes. (See, e.g. , Bril, WS et al. (2006) Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg(593) to Cys substitution. Thromb Haemost 95, 341-347; Homanics, GE et al. (2006) Production and characterization of murine models of classic and intermediate maple syrup urine disease.BMC Med Genet 7, 33; Jamsai, D. et al. (2006) A humanized BAC transgenic/knockout mouse model for HbE/ beta-thalassemia.Genomics 88(3):309-15; Pan, Q. et al. (2006) Different role for mouse and human CD3delta/epsilon heterodimer in preT cell receptor (preTCR) function: human CD3delta/epsilon heterodimer restores the defective preTCR function in CD3gamma- and CD3gammadelta-deficient mice.Mol Immunol 43, 1741-1750). However, this effort has been hampered by size restrictions; Conventional knockout techniques have not been sufficient to directly replace a large mouse gene against its large human genome. The simple approach of direct homology replacement is rarely attempted because of technical difficulties, where the endogenous mouse gene is replaced directly by the human relative gene at the same exact genetic location of the mouse gene (ie, in the endogenous mouse site). To date, efforts in direct replacement have entailed elaborate and laborious procedures, thus limiting the length of genetic material that can be processed and the accuracy with which it can be manipulated.

Human immunoglobulin transgenes introduced exogenously rearrange precursor B-cells in mice (Alt, FW, Blackwell, TK, and Yancopoulos, GD (1985). Immunoglobulin genes in transgenic mice. Trends Genet 1, 231-236 ). This finding was used by genetically engineered mice using a knockout-plus-transformation approach to express human antibodies (Green, LL et al. (1994) Antigen-specific human monoclonal antibodies from mice engineered with human Ig. heavy and light chain YACs.Nat Genet 7, 13-21; Lonberg, N. (2005).Human antibodies from transgenic animals.Nat Biotechnol 23, 1117-1125; Lonberg, N. et al. (1994) Antigen-specific human antibodies from mice comprising four distinct genetic modifications.Nature 368, 856-859; Jakobovits, A. et al. (2007) From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice.Nat Biotechnol 25, 1134-1143) . Endogenous mouse immunoglobulin heavy chain and κ light chain loci are inactivated in these mice by targeted deletion of a small but significant portion of each endogenous locus and then as randomly integrated macrotransgenes as described above, or human as minichromosomes. Immunoglobulin loci were introduced (Tomizuka, K. et al. (2000) Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies.Proc Natl Acad Sci USA 97 , 722-727). These mice have shown significant advances in genetic manipulation; Fully human monoclonal antibodies isolated from them have obtained promising therapeutic potential for targeting various human diseases (Gibson, TB et al. (2006) Randomized phase III trial results of panitumumab, a fully human anti-epidermal growth factor receptor). monoclonal antibody, in metastatic colorectal cancer.Clin Colorectal Cancer 6, 29-31; Jakobovits et al., 2007; Kim, YH et al. (2007) Clinical efficacy of zanolimumab (HuMax-CD4): two Phase II studies in refractory cutaneous T-cell lymphoma.Blood 109(11):4655-62; Lonberg, 2005; Maker, AV et al. (2005) Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: a phase I/II study.Ann Surg Oncol 12, 1005-1016; McClung, MR, Lewiecki, EM et al. (2006) Denosumab in postmenopausal women with low bone mineral density.N Engl J Med 354, 821-831). However, as discussed above, these mice show compromised B cell development and immunodeficiency when compared to wild-type mice. These problems support violent humoral responses and, as a result, potentially limit the ability of mice to make fully human antibodies against some antigens. Deficiencies may be due to: (1) insufficient functionality due to random introduction of human immunoglobulin transgenes and inaccurate expression obtained due to lack of upstream and downstream control elements (Garrett, FE et al. ( 2005) Chromatin architecture near a potential 3'end of the igh locus involves modular regulation of histone modifications during B-Cell development and in vivo occupancy at CTCF sites.Mol Cell Biol 25, 1511-1525; Manis, JP et al. (2003 ) Elucidation of a downstream boundary of the 3'IgH regulatory region.Mol Immunol 39, 753-760; Pawlitzky, I. et al. (2006) Identification of a candidate regulatory element within the 5'flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A. J Immunol 176, 6839-6851); (2) Indistinct interspecies interaction between the human constant domain of the B-cell receptor signal processing complex on the cell surface and the mouse component, which may impart signal processing necessary for normal maturation, proliferation and survival of B cells ( Hombach, J. et al. (1990) Molecular components of the B-cell antigen receptor complex of the IgM class. Nature 343, 760-762); And (3) insufficient interspecies interaction between soluble human immunoglobulins and mouse Fc receptors that can reduce affinity selection (Rao, SP et al. (2002) Differential expression of the inhibitory IgG Fc receptor FcgammaRIIB on germinal center cells: implications for selection of high-affinity B cells.J Immunol 169, 1859-1868) and immunoglobulin serum concentration (Brambell, FW et al. (1964).A Theoretical Model of Gamma-Globulin Catabolism.Nature 203, 1352-1354; Junghans, RP, and Anderson, CL (1996).The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor.Proc Natl Acad Sci USA 93, 5512-5516; Rao et al. , 2002; Hjelm, F. et al. (2006) Antibody-mediated regulation of the immune response.Scand J Immunol 64, 177-184; Nimmerjahn, F., and Ravetch, JV (2007).Fc-receptors as regulators of immunity.Adv Immunol 96 , 179-204). These deficiencies can be corrected by in situ humanization of only the variable regions of the mouse immunoglobulin site within its natural site at the endogenous heavy and light chain sites. This effectively results in mice making "reverse chimera" (ie, human V: mouse C) antibodies that can be selected and interacted normally with a mouse environment based on the mouse constant region they possess. Additionally, such reverse chimeric antibodies can be readily reconfigured to fully human antibodies for therapeutic purposes.

Genetically modified animals that contain a replacement at an endogenous immunoglobulin heavy chain site with a heterologous (e.g., from another species) immunoglobulin sequence may be treated with a replacement at the endogenous immunoglobulin light chain site or with an immunoglobulin light chain transgene (e.g., chimeric immunoglobulin). Light chain transgenes or intact human intact mice, etc.). The species from which the heterologous immunoglobulin heavy chain sequence is derived can be significantly different from the immunoglobulin light chain sequence used in the immunoglobulin light chain sequence replacement or the immunoglobulin light chain transgene.

Immunoglobulin variable region nucleic acid sequences such as V, D, and/or J segments are obtained from human or non-human animals in various embodiments. Non-human animals suitable for providing V, D, and/or J segments are, for example, cartilage fish such as bony fish, sharks and stingrays, non-humans such as amphibians, reptiles, mammals and birds (e.g. chickens). Includes animals. Non-human animals include, for example, mammals. Mammals include, for example, non-human primates, goats, sheep, pigs, dogs, cattle (e.g. cows, bulls, buffaloes), deer, camels, ferrets and rodents and non-human primates (e.g. chimpanzees, orangutans, Gorilla, marmoset, rhesus monkey baboon). Suitable non-human animals are selected from the rodent family, including rats, mice, and hamsters. In one embodiment, the non-human animal is a mouse. As will be clear from the context, various non-human animals can be used as a source of variable domain or variable region gene segments (eg, sharks, rays, mammals (eg, camels, rodents, such as mice and rats).

According to the context, non-human animals are also used as a source of variable sequences or constant region sequences used in connection with segments, e.g., rodent constant sequences are human or non-human variable sequences (e.g., rodents, e.g., It can be used in a transgene operably linked to a human or non-human primate variable sequence, constant sequence) operably linked to a mouse or rat or hamster. Thus, in various embodiments, the human V, D, and/or J segments are operably linked to a rodent (eg, mouse or rat or hamster) constant region gene sequence. In certain embodiments, the human V, D, and/or J segment (or one or more rearranged VDJ or VJ genes) is a mouse, rat, or hamster in a transgene integrated at a site other than the endogenous immunoglobulin site. Operably linked or fused to the constant region gene sequence.

In a specific embodiment, there is provided a mouse comprising replacement of the _{V H} , D _H , and J _H gene segments at an endogenous immunoglobulin heavy chain locus having _{one or more human V H} , D _H , and J _{H segments, wherein one or more} Human V _H , D _H , and J _H segments are operably linked to endogenous immunoglobulin heavy chain constant genes; Mice contain transgenes at sites other than endogenous immunoglobulin sites, transgenes comprising unrearranged or rearranged human V _L and human J _L segments operably linked to mouse or rat or human constant regions. .

In a specific embodiment, a mouse is provided comprising the insertion of _{one or more human V H} , D _H and J _H gene segments at an endogenous immunoglobulin heavy chain locus. In one embodiment, the insertion is upstream of an endogenous immunoglobulin heavy chain constant gene; In one embodiment, the insertion is downstream of an endogenous variable (V) gene segment; In one embodiment, the insertion is downstream of an endogenous diversity (D) gene segment; In one embodiment, the insertion is downstream of an endogenous binding (J) gene segment. In various embodiments, the insertion is such that one or more human V _H , D _H and J _H gene segments are positioned with an operative linkage to one or more endogenous heavy chain constant genes.

A method is described that maintains the ability of mice to produce offspring while performing a large in situ genetic replacement of a mouse germline immunoglobulin variable locus in a human germline immunoglobulin variable locus. Specifically, it is described to make exact replacement of 6 megabases of both mouse heavy chain and κ light chain immunoglobulin variable loci with human counterparts while leaving the mouse constant region that is intact. As a result, mice made an accurate replacement of the entire germline immunoglobulin variable repertoire of mice equivalent to the human germline immunoglobulin variable sequence, while maintaining the mouse constant region. Human variable regions are linked to mouse constant regions to form chimeric human-mouse immunoglobulin sites that are rearranged and expressed at physiologically appropriate levels. The expressed antibodies are “reverse chimeric”, ie they include human variable region sequences and mouse constant region sequences. These mice with humanized immunoglobulin variable regions expressing antibodies with human variable regions and mouse constant regions are called VELOCIMMUNE® mice.

VELOCIMMUNE® humanized mice exhibit a fully functional humoral immune system that is essentially indistinguishable from wild-type mice. They represent normal cell populations at all stages of B cell development. They represent a normal lymphoid organ morphology. The antibody sequence of VELOCIMMUNE (registered trademark) mice shows normal V(D)J rearrangement and normal somatic hypermutation frequency. The antibody population in these mice reflects the isotype distribution resulting from normal classification shift (eg, normal isotype cis-shift). Immunizing VELOCIMMUNE® mice results in a strong humoral immune response that creates a large, diverse antibody repertoire with human immunoglobulin variable domains suitable as therapeutic candidates. This platform provides a rich source of naturally affinity-matured human immunoglobulin variable region sequences to make pharmaceutically acceptable antibodies and other antigen-binding proteins.

There is an exact replacement of the mouse immunoglobulin variable sequence with the human immunoglobulin variable sequence that makes VELOCIMMUNE® mice. In addition, even the exact replacement of the endogenous mouse immunoglobulin sequence at the heavy and light chain sites with equivalent immunoglobulin sequences by a very large range of sequential recombination of the human immunoglobulin sequence is specific due to the various evolution of immunoglobulin sites between mice and humans. It can exist as a challenge. For example, intergenic sequences interspersed within immunoglobulin sites are not identical between mice and humans, but in some cases may not be functionally identical. Differences in its immunoglobulin loci between mice and humans can still lead to abnormalities in humanized mice, especially when certain portions of the endogenous mouse immunoglobulin heavy chain loci are humanized or manipulated. Some modifications in the mouse immunoglobulin heavy chain locus are detrimental. Harmful modifications can include, for example, loss of the ability of the modified mice to mate or produce offspring. In various embodiments, engineering the human immunoglobulin sequence in the genome of the mouse includes a method of maintaining a detrimental endogenous sequence when absent in the modified mouse strain. Exemplary detrimental effects may include inability to proliferate the modified strain, loss of function of essential genes, inability to express the polypeptide, and the like. These detrimental effects can be directly or indirectly related to genetically engineered modifications into the genome of the mouse.

6 megabases of accurate, large-scale in situ replacement _{of the variable regions (V H} -D _H -J _H and Vκ-Jκ) of mouse heavy and light chain immunoglobulin sites with corresponding 1.4 megabase human genomic sequences was performed. On the other hand, flanking mouse sequences are deviated within a defective and functional hybrid site, including all mouse constant chain genes and site transcription control regions (FIGS. 1A and 1B). Specifically, the human V _H , D _H , J _H , Vκ and Jκ gene sequences were made of 13 chimeric BAC targeting vectors containing overlapping fragments of human germline variable sites into mouse ES cells using VELOCKGENE (registered trademark) genetic engineering technology. It was introduced through stepwise insertion (eg , US Pat. No. 6,586,251 and Valenzuela, DM et al. (2003). High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21, 652-659 ] Reference).

Humanization of the mouse immunoglobulin gene represents the largest genetic modification to the mouse genome to date. While previous efforts with randomly integrated human immunoglobulin transgenes have met some success (discussed above), direct replacement of the mouse immunoglobulin gene with its human counterpart has resulted in the efficient use of fully-human antibodies in other normal mice. It dramatically increases the efficiency that can be generated. Additionally, these mice display a dramatically increased diversity of fully-human antibodies that can be obtained after immunization with virtually any antigen compared to mice containing the impaired endogenous site and the fully human antibody transgene. The various types of replaced, humanized sites represent completely normal levels of mature and immature B cells, in contrast to mice carrying randomly integrated human transgenes, indicating significantly reduced B cells at various stages of differentiation. Efforts to increase the number of human gene segments in human transgenic mice reduced this defect, but the expanded immunoglobulin repertoire did not have a wholly corrected reduction in the B cell population compared to wild-type mice.

Immunity observed in mice with immunoglobulin sites (i.e., VELOCIMMUNE® mice) in which nearby wild-type humoral immune function has been replaced, but not encountered in some approaches using randomly integrated transgenes. There are other challenges encountered when using direct replacement of globulin. Differences in the genetic composition of immunoglobulin loci between mice and humans have led to the discovery of sequences that are beneficial for the proliferation of mice carrying the replaced immunoglobulin gene segments. Specifically, the mouse ADAM gene located within the endogenous immunoglobulin site is optimally present in mice with the replaced immunoglobulin site because of its role in fertility.

Genomic location and function of mouse ADAM6

Male mice lacking the ability to any functional ADAM6 protein surprisingly display a defect in the ability of mice to mate and produce offspring. Mice lack the ability to express functional ADAM6 protein because of the replacement of all or substantially all mouse immunoglobulin variable region gene segments with human variable region gene segments. The loss of ADAM6 function results because it is located within the region of the endogenous mouse immunoglobulin heavy chain variable region locus proximal to the _{3'end of the V H} gene segment locus, which is upstream of the _{D H gene segment.} In order to breed homozygous mice for replacement of all or substantially all endogenous mouse heavy chain variable gene segments with human heavy chain variable gene segments, a cumbersome approach to prepare males and females awaiting a homozygous and productive mating respectively for replacement is a cumbersome approach. It is common. Successful litters are less frequent and less frequent. Instead, males that are heterozygous for the replacement are used for mating with females that are homozygous for the replacement to create offspring that are heterozygous for the replacement, and then the homozygous mice are crossed from it. The inventors have determined that a possible cause of loss of fertility in male mice is the absence of homozygous male mice of the functional ADAM6 protein.

In various aspects, male mice containing an impaired (ie, non-functional or slightly functional) ADAM6 gene exhibit a reduction or elimination of fertility. Since the ADAM6 gene in mice (and other rodents) is located within the immunoglobulin heavy chain locus, we have made various modifications to propagate mice, or to create and maintain strains of mice containing the replaced immunoglobulin heavy chain loci. It was determined that a mating or propagation scheme was used. The low fertility, or nonfertility, of male mice homozygous for the replacement of endogenous immunoglobulin heavy chain variable loci makes it difficult to maintain this modification in mouse strains. In various embodiments, maintaining the strain includes avoiding the nonfertility problems presented by male mice homozygous for replacement.

In one aspect, a method for maintaining a strain of a mouse as described herein is provided. The strain of the mouse need not contain an ectopic ADAM6 sequence, and in various embodiments the strain of the mouse is homozygous or heterozygous for knockout of ADAM6 (eg, functional knockout).

Mouse strains contain modifications of endogenous immunoglobulin heavy chain sites that result in a decrease or loss of fertility in male mice. In one embodiment, the modification comprises deletion of a regulatory region and/or a coding region of the ADAM6 gene. In a specific embodiment, the modification comprises modification of the endogenous ADAM6 gene (regulatory and/or coding region) that reduces or eliminates the fertility of the male mouse comprising the modification; In a specific embodiment, the modification reduces or eliminates the fertility of male mice homozygous for the modification.

In one embodiment, the mouse strain is homozygous or heterozygous for knockout (eg, functional knockout) or deletion of the ADAM6 gene.

In one embodiment, the mouse strain is to isolate cells from mice that are homozygous or heterozygous for modification, use donor cells in host embryos, conceive host embryos and donor cells in surrogate mothers, and genes. It is maintained by obtaining a surrogate mother from the surrogate mother, including the transformation. In one embodiment, the donor cell is an ES cell. In one embodiment, the donor cell is a pluripotent cell, such as an induced pluripotent cell.

In one embodiment, the mouse strain is to isolate the nucleic acid sequence comprising the modification from the mouse homozygous or heterozygous for the modification, and to introduce the nucleic acid sequence into the host nucleus, and in a suitable animal the nucleic acid sequence and It is maintained by conceiving cells containing the host nucleus. In one embodiment, the nucleic acid sequence is introduced into the host oocyte embryo.

In one embodiment, the mouse strain is to isolate the nucleus from a mouse that is homozygous or heterozygous for the modification, and introduces the nucleus into the host cell, and conceives the nucleus and host cell in a suitable animal for modification. Is maintained by obtaining a homozygous or heterozygous offspring.

In one embodiment, the mouse strain is in vitro fertilization of a male mouse (wild type, homozygous for modification, or heterozygous for modification) using sperm from a male mouse containing the genetic modification. : Maintained by using IVF). In one embodiment, the male mouse is heterozygous for genetic modification. In one embodiment, the male mouse is homozygous for genetic modification.

In one embodiment, the mouse strain is to cross-cross a male mouse heterozygous for genetic modification with a female mouse to obtain offspring comprising the genetic modification, to identify male and female offspring comprising the genetic modification, and crossbreeding. Is maintained by using males that are heterozygous for the genetic modification in conjunction with females that are wild-type, homozygous, or heterozygous for the genetic modification to obtain offspring comprising the genetic modification. In one embodiment, the step of crossing a male heterozygous for genetic modification with a wild-type female, a female heterozygous for the genetic modification, or a female homozygous for the genetic modification is to maintain the genetic modification in the mouse strain. It repeats.

In one aspect, a method for maintaining a mouse strain comprising replacing the endogenous immunoglobulin heavy chain variable locus with one or more human immunoglobulin heavy chain sequences comprising crossing the mouse strain to make a heterozygous male mouse While provided, heterozygous male mice are bred to maintain genetic modification in the strain. In a specific embodiment, the strain is not maintained by any crossing with a wild-type female of a homozygous male, a female homozygous or heterozygous for the genetic modification.

The ADAM6 protein is a member of the ADAM family of proteins, where ADAM is an acronym for A disintegrin and metalloprotease. The ADAM family of proteins is large and diverse, and has a variety of functions, including cell adhesion. Some members of the ADAM family are involved in spermatogenesis and fertilization. For example, ADAM2 encodes a subunit of the protein fertilin, which is involved in sperm-oocyte interactions. ADAM3, or cyritestin, appears to be required for sperm binding to the zona pellucida. Absence of either ADAM2 or ADAM3 results in nonfertility. ADAM2, ADAM3 and ADAM6 were hypothesized to form a complex on the surface of mouse sperm cells.

The human ADAM6 gene normally found between the liver V _H gene segments V _H 1-2 and V _H 6-1 appears to be a pseudogene (FIG. 12 ). In mice, _{there are two genes-ADAM6a and ADAM6b- found in the intergenic region between the mouse V H} and D _H gene segments, and in mice, the ADAM6a and ADAM6b genes are transcribed opposite to the genes of the surrounding immunoglobulin gene segments. Oriented to the orientation (Figure 12). In mice, a functional ADAM6 site is clearly required for normal fertilization. The functional ADAM6 site or sequence then refers to the complementary or salvable ADAM6 site or sequence, with dramatically reduced fertilization in male mice with a missing or non-functional endogenous ADAM6 site.

The location of the intergenic sequence in mice encoding ADAM6a and ADAM6b provides an intergenic sequence that is susceptible to modification when modifying the endogenous mouse heavy chain. When the V _H gene segment is deleted or replaced, or when the D _H gene segment is deleted or replaced, there is a high likelihood that the resulting mice will show a serious deletion in fertility. To compensate for the deletion, mice are modified to contain a nucleotide sequence encoding a protein that will compensate for the loss of ADAM6 activity due to modification of the endogenous mouse ADAM6 site. In various embodiments, the complementary nucleotide sequence is one that encodes mouse ADAM6a, mouse ADAM6b, or a homolog or ortholog or a functional fragment thereof that rescues the fertility defect. Alternatively, suitable methods for conserving the endogenous ADAM6 site can be used, while providing an endogenous immunoglobulin heavy chain sequence flanking the mouse ADAM6 site that cannot be rearranged to encode a functional endogenous heavy chain variable region. An exemplary alternative method involves the manipulation of large portions of the mouse chromosome to locate endogenous immunoglobulin heavy chain variable region sites, in which they are rearranged to encode a functional heavy chain variable region operably linked to an endogenous heavy chain constant gene. Can't be In various embodiments, the method comprises inversion and/or translocation of a mouse chromosome fragment containing an endogenous immunoglobulin heavy chain gene segment.

The nucleotide sequence that rescues fertility can be located at any suitable position. It can be located in the intergenic region, or at any suitable location in the genome (ie, ectopic). In one embodiment, the nucleotide sequence can be introduced into a transgene that randomly integrates into the mouse genome. In one embodiment, the sequence may be maintained episomally, ie on a separate nucleic acid than a mouse chromosome. Suitable positions are transcriptionally tolerant or active positions such as ROSA26 site (Zambrowicz et al., 1997, PNAS USA 94:3789-3794), BT-5 site (Michael et al. , 1999, Mech. Dev. 85: 35-47), or Oct4 site (Wallace et al. , 2000, Nucleic Acids Res. 28:1455-1464). Targeting nucleotide sequences to transcriptionally active sites is described, for example, in US Pat. No. 7,473,557, incorporated herein by reference.

Alternatively, the nucleotide sequence that rescues fertility can be associated with an inducible promoter to allow for optimal expression in appropriate cells and/or tissues, such as regenerative tissues. Exemplary inducible promoters include promoters activated by physical (eg, heat shock promoter) and/or chemical means (eg , IPTG or tetracycline).

Additionally, expression of the nucleotide sequence can be linked to other genes to achieve expression at specific stages of development or within specific tissues. Such expression can be achieved by locating a nucleotide sequence operably linked to the promoter of the gene expressed at a specific stage of development. For example, an immunoglobulin sequence from one species engineered into the genome of the host species is placed in an operative linkage with the promoter sequence of the CD19 gene (B cell specific gene) from the host species. When immunoglobulins are expressed, B cell-specific expression is achieved at the correct stage of development.

Another way to achieve strong expression of the inserted nucleotide sequence is to use a constitutive promoter. Exemplary constitutive promoters include SV40, CMV, UBC, EF1A, PGK and CAGG. In a similar manner, the desired nucleotide sequence is placed in an operative linkage with the selected constitutive promoter, which provides a high level of expression of the protein(s) encoded by the nucleotide sequence.

The term “ectopic” is intended to include movement or placement at a location that does not normally occur in nature (eg, placement of a nucleic acid sequence at a location where the nucleic acid sequence is not the same location as found in wild-type mice). The term is used in various embodiments in the sense of its purpose outside of its normal or appropriate position. For example, the phrase “encoding an ectopic nucleotide sequence” refers to a nucleotide sequence that appears at a location that does not normally occur in a mouse. For example, in the case of an ectopic nucleotide sequence encoding a mouse ADAM6 protein (or an ortholog or homolog or fragment thereof that provides the same or similar fertility advantage in male mice), the sequence is more than that normally found in wild-type mice. It can be located at different locations in the mouse genome. In this case, a new sequence junction of the mouse sequence will be made by placing the sequence at a different location in the genome of the mouse than in the wild-type mouse. The functional homolog or ortholog of mouse ADAM6 is ^{a sequence conferring the loss of fertility observed in ADAM6 −/−} mice (eg, loss of the ability of male mice to produce offspring by mating). Functional homologs or orthologs have at least about 89% homology or more, such as 99% homology to the amino acid sequence of ADAM6a and/or to the amino acid sequence of ADAM6b, or deletion or knockout of ADAM6a and/or ADAM6b. It includes a protein that rescues the ability of mice with genotypes to mate successfully.

The ectopic location can be anywhere (e.g. , such as random insertion of a transgene containing the mouse ADAM6 sequence), or at a location close to its location, e.g., in a wild-type mouse (e.g., a modified endogenous mouse immunoglobulin site. However, it may be either upstream or downstream of its native position, such as within a modified immunoglobulin site, but between different gene segments or at different positions within the mouse VD intergenic sequence). An example of an ectopic site is to provide a peripheral endogenous heavy chain gene segment that can be rearranged to encode a functional heavy chain containing an endogenous heavy chain constant region while maintaining the position normally found in wild-type mice within the endogenous immunoglobulin heavy chain locus. . In this example, this can be achieved by inversion of the chromosome fragment containing the endogenous immunoglobulin heavy chain variable site, such as by using an engineered site specific recombination site located at a position flanking the variable region site. Thus, upon recombination, the endogenous heavy chain variable region locus is located at a large distance from the endogenous heavy chain constant region gene, thereby preventing rearrangement to encode a functional heavy chain containing the endogenous heavy chain constant region. Other exemplary methods of achieving functional silencing of the endogenous immunoglobulin heavy chain variable locus while maintaining the functional ADAM6 locus will be apparent to those skilled in the art by reading the present disclosure and/or in combination with methods known in the art. By this arrangement of the endogenous heavy chain locus, the endogenous ADAM6 gene is maintained and the endogenous immunoglobulin heavy chain locus is functionally silenced.

Another example of an ectopic configuration is a configuration within a humanized immunoglobulin heavy chain site. For example, a mouse comprising a replacement of one or more endogenous V _H gene segments with a human V _H gene segment can be genetically engineered to have a mouse ADAM6 sequence located within a sequence containing the _{human V H gene segment, wherein the replacement is endogenous.} Remove the ADAM6 sequence. The resulting modification creates a (ectopic) mouse ADAM6 sequence within the human gene sequence, and the (ectopic) placement of the mouse ADAM6 sequence within the human gene sequence may be close to the location of the human ADAM6 pseudogene (i.e. between the two V segments) or Or close to the location of the mouse ADAM6 sequence (ie, within the VD intergenic region). The resulting sequence junction, which is made by the association of a human gene sequence (e.g. , immunoglobulin gene sequence) or adjacent (ectopic) mouse ADAM6 sequence in the germline of the mouse, is novel relative to the same or similar position in the genome of the wild-type mouse.

In various embodiments, a non-human animal is provided that is free of ADAM6 or an ortholog or homolog thereof, wherein the lack provides a non-human animal that is non-fertile, or substantially reduces the fertility of the non-human animal. In various embodiments, the lack of ADAM6 or an ortholog or homolog thereof is due to a modification of the endogenous immunoglobulin heavy chain locus. A substantial decrease in fertility is, for example, a decrease in fertility (eg, mating frequency, litter, offspring per year, etc.) of about 50%, 60%, 70%, 80%, 90% or 95% or more. In various embodiments, the non-human animal is supplemented with a mouse ADAM6 gene or ortholog or homolog or a functional fragment thereof that is functional in the male of the non-human animal, but supplemented ADAM6 gene or ortholog or homolog or functional thereof. Fragments rescue the decrease in fertility in whole or in a substantial part. The salvage of fertility in a substantial part is, for example, restoration of fertility, so non-human animals are at least 70%, 80% relative to the heavy chain locus of the unmodified (i.e., no modification to the ADAM6 gene or its ortholog or homolog). Or 90% or more of fertility.

The sequence imparted to a genetically modified animal (i.e., an animal without functional ADAM6 or an ortholog or homolog thereof due to modification of an immunoglobulin heavy chain locus) is, in various embodiments, the ADAM6 gene or an ortholog or homolog thereof. It is selected from For example, in mice, the loss of ADAM6 function is rescued, in one embodiment, by adding the mouse ADAM6 gene. In one embodiment, the loss of ADAM6 function in the mouse is by adding an ortholog or homolog of a closely related species to a mouse, such as a rodent, such as a different strain or species of mouse, any species of rat, rodent. Saved; Addition of orthologs or homologs to mice rescues loss of fertility due to loss of ADAM6 function or loss of ADAM6 gene. Orthologs and homologs from other species are, in various embodiments, selected from phylogenetically related species, and in various embodiments, about 80% or more, 85% or more, 90% or more, 95% or more, 96 % Or more or 97% or more and represents a percent identity with endogenous ADAM6 (or ortholog); ADAM6-related or (in non-mouse) ADAM6 ortholog-related losses of fertility are rescued. For example, in genetically modified male rats without ADAM6 function (e.g., rats with human immunoglobulin heavy chain variable regions in rat immunoglobulin heavy chain regions, or with endogenous immunoglobulin heavy chain variable regions replaced by knockout), fertile in rats Sex loss is rescued by the addition of rat ADAM6 or, in certain embodiments, an ortholog of rat ADAM6 (eg, from another strain or species, or, in one embodiment, from a mouse).

Thus, in various embodiments, a genetic modification showing a decrease in fertility or no fertility due to modification of the nucleic acid sequence encoding the ADAM6 protein (or an ortholog or homolog thereof) or regulatory region operably linked to the nucleic acid sequence. The resulting animal comprises a nucleic acid sequence that compensates for or recovers the loss of fertility when the nucleic acid sequence that compensates for or recovers from a different strain of the same species or from a phylogenetically related species. In various embodiments, the complementing nucleic acid sequence is an ADAM6 ortholog or homolog or a functional fragment thereof. In various embodiments, complementing the ADAM6 ortholog or homolog or functional fragment thereof is derived from a non-human animal closely related to a genetically modified animal having fertility binding. For example, if the genetically modified animal is a mouse of a specific strain, an ADAM6 ortholog or homolog or a functional fragment thereof can be obtained from a mouse of another strain or a mouse of a related species. In one embodiment, when the genetically modified animal comprising the fertility defect is of the order rodent, the ADAM6 ortholog or homolog or functional fragment thereof is derived from another animal of the order rodent. In one embodiment, the genetically modified animal comprising a fertility defect is a suborder of rats (e.g., flying rats, flying pigs, mouse-like hamsters, hamsters, New World rats and mice, voles, true mice and rats. , Gerbils rat, spiny pouch, mane rat, climbing mouse, rock mouse, white-tailed rat, Madagascar rat and mouse, spiny winter rat, wolfram, bamboo rat, braid mole), and ADAM6 ortholog or homolog or Its functional fragment is selected from animals of the order rodent or order rodent.

In one embodiment, the genetically modified animal is derived from the murine family, and the ADAM6 ortholog or homolog or functional fragment thereof is derived from the murine family. In one embodiment, the genetically modified animal is derived from the murine family, and the ADAM6 ortholog or homolog or functional fragment thereof is derived from the murine family.

In one embodiment, it is a genetically modified animal rodent. In one embodiment, the rodent is selected from the murine family, and the ADAM6 ortholog or homolog is derived from a different species within the murine family. In one embodiment, the genetically modified animal is Calomiscus family (e.g., mouse-like hamster), silkworm family (e.g., hamsters, New World rats and mice, cheeks), rodents (true mice and rats, gerbils Rats, spiny pouch rats, cypress rats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Madagascar rats and mice), spiny winter-sleeping rats (e.g., spiny winter-sleeping rats), and bamboo rats (e.g. , Musculus rats, bamboo rats, and braided moles); The ADAM6 ortholog or homolog is selected from different species of the same family. In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Rataceae), and the ADAM6 ortholog or homologue is selected from gerbils, spiny pouch, or cervical rats. In one embodiment, the genetically modified mouse is from a member of the murine family, and the ADAM6 ortholog or homolog is from a different species of the murine family. In a specific embodiment, the genetically modified rodent is a mouse of the order Musculaceae, and the ADAM6 ortholog or homolog is derived from a rat of the murine family, a gerbil, a thorny sac, or a rat.

In various embodiments, one or more rodent ADAM6 orthologs or homologs or functional fragments thereof of rodents in the family are genetically modified rodents of the same family without ADAM6 orthologs or homologs (e.g. World rats and mice, cheeks); regains fertility for rodents (e.g., true mice and rats, gerbils, spiny pouch mice, chinensis rats)).

In various embodiments, the ADAM6 ortholog, homologue, and fragment thereof is a genetically modified male non-human animal in which the ortholog, homologue, or fragment lacks ADAM6 activity (e.g., knockout of ADAM6 or an ortholog thereof. Rodents, including, for example, mice or rats) are evaluated for functionality by ascertaining whether they recover fertility. In various embodiments, functionality is defined as the sperm ability of a genetically modified animal without endogenous ADAM6 or its orthologs or homologs to move the fallopian tube or fertilize an egg of the same species of the genetically modified animal.

In various aspects, an endogenous heavy chain variable region site containing an ectopic nucleotide sequence encoding a protein that confers similar fertility advantages to mouse ADAM6 (e.g., functional orthologs or homologs or fragments thereof in male mice) or Mice can be made that contain deletions or replacements of parts of them. Ectopic nucleotide sequences encode proteins that are different mouse strains or different species, such as ADAM6 homologs or orthologs (or fragments thereof) of different rodent species, and have advantages in fertility, such as increased number of pups over a specified time period. And/or a nucleotide sequence that confers the ability of the sperm cells of the male mouse to traverse through the mouse oviduct to fertilize the mouse ovary.

In one embodiment, ADAM6 is a homologue or ortholog that is at least 89% to 99% identical to mouse ADAM6 protein (eg, at least 89% to 99% identical to mouse ADAM6a or mouse ADAM6b). In one embodiment, the ectopic nucleotide sequence encodes one or more proteins independently selected from a protein that is at least 89% identical to mouse ADAM6a, a protein that is at least 89% identical to mouse ADAM6b, and combinations thereof. In one embodiment, the homologue or ortholog is a rat, hamster, mouse, or guinea pig protein that is at least about 89% identical or modified to mouse ADAM6a and/or mouse ADAM6b. In one embodiment, the homologue or ortholog is the same or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 for mouse ADAM6a and/or mouse ADAM6b. % Or 99% the same.

In one aspect, a non-human animal is provided, wherein the non-human animal (a) inserts one or more human Vλ and Jλ gene segments upstream of the non-human immunoglobulin light chain constant region, (b) non-human immunity _{Insertion of one or more human V H} , one or more human D _H and one or more human J _H gene segments upstream of the globulin heavy chain constant region, and (c) a nucleotide sequence encoding an ADAM6 protein or a functional fragment thereof. In one embodiment, the non-human heavy and/or light chain constant regions are rodent constant regions (eg, selected from mouse, rat or hamster constant regions). In one embodiment, the non-human light chain constant region is a rodent constant region. In a specific embodiment, the light chain constant region is a mouse Cκ or rat Cκ region. In a specific embodiment, the light chain constant region is a mouse Cλ or rat Cκ region. Suitable non-human animal is a rodent, for example, include mouse, rat and hamster. In one embodiment, the rodent is a mouse or rat.

In one embodiment, the non-human animal comprises at least 12 to at least 40 human Vλ gene segments and at least one human Jλ gene segment. In a specific embodiment, the non-human animal comprises 12 human Vλ gene segments and at least one human Jλ gene segment. In a specific embodiment, the non-human animal comprises 28 human Vλ gene segments and at least one human Jλ gene segment. In one embodiment, the non-human animal comprises 40 human Vλ gene segments and at least one human Jλ gene segment. In various embodiments, at least one human Jλ gene segment is selected from Jλ1, Jλ2, Jλ3 and Jλ7. In a specific embodiment, the non-human animal comprises at least 4 human Jλ gene segments. In one embodiment, the at least four human Jλ gene segments comprise at least Jλ1, Jλ2, Jλ3 and Jλ7.

In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof is ectopic in a non-human animal. In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof (functional in non-human animals) is at the same position compared to the wild-type non-human ADAM6 site. In one embodiment, the non-human animal is a mouse and the nucleotide sequence encodes a mouse ADAM6 protein or a functional fragment thereof, and is present at an ectopic location in the genome of the non-human animal. In one embodiment, the non-human animal is a mouse and the nucleotide sequence encodes a mouse ADAM6 protein or a functional fragment thereof, and is present in an immunoglobulin gene segment. In a specific embodiment, the immunoglobulin gene segment is a heavy chain gene segment. In one embodiment, the heavy chain gene segment is human. In one embodiment, the heavy chain gene segment is an endogenous heavy chain gene segment from a non-human animal. In one embodiment, the mouse comprises an ectopic contiguous sequence comprising one or more endogenously unrearranged heavy chain gene segments, and the ADAM6 sequence is within an ectopic contiguous sequence.

In one embodiment, the non-human animal _{lacks endogenous immunoglobulin V L} and/or J _L gene segments at the endogenous immunoglobulin light chain site. In one embodiment, the non-human animal comprises _{endogenous immunoglobulin V L} and/or J _L gene segments that cannot be rearranged to form an _{immunoglobulin V L domain in the non-human animal.} In one embodiment, all or substantially all of the endogenous immunoglobulin VK and JK gene segments are replaced with one or more human Vλ and Jλ gene segments. In one embodiment, all or substantially all of the endogenous immunoglobulin Vλ and Jλ gene segments are replaced with one or more human Vλ and Jλ gene segments. In one embodiment, all or substantially all of the endogenous immunoglobulin V _L and J _L gene segments are intact in a non-human animal, and the non-human animal has endogenous immunoglobulin V _L and/or J _L gene segments and endogenous At least one human Vλ gene segment and at least one human Jλ gene segment inserted between the immunoglobulin light chain constant regions. _{In a specific embodiment, intact endogenous immunoglobulin V L} and J _L gene segments are provided that cannot be rearranged to form _{the V L} domain of an antibody in a non-human animal. In various embodiments, the endogenous immunoglobulin light chain site of the non-human animal is an immunoglobulin κ light chain site. In various embodiments, the endogenous immunoglobulin light chain site of the non-human animal is an immunoglobulin λ light chain site. In various embodiments, the endogenous immunoglobulin V _L and J _L gene segments are VK and JK gene segments. In various embodiments, the endogenous immunoglobulin V _L and J _L gene segments are Vλ and Jλ gene segments.

In one embodiment, the non-human animal further comprises a human Vκ-Jκ intergenic region from a human κ light chain locus, wherein the human Vκ-Jκ intergenic region is contiguous with one or more human Vλ and Jλ gene segments. In a specific embodiment, the human Vκ-Jκ intergenic region is located between a human Vλ gene segment and a human Jλ gene segment.

In one aspect, cells and/or tissues derived from a non-human animal as described herein are provided, wherein the cells and/or tissues are (a) one or more of the upstream of the non-human immunoglobulin light chain constant region. Insertion of human Vλ and Jλ gene segments, (b) insertion of one or more human V _H , one or more human D _H and one or more human J _H gene segments upstream of the non-human immunoglobulin heavy chain constant region, and (c) ADAM6 It includes a nucleotide sequence encoding a protein or a functional fragment thereof. In one embodiment, the non-human heavy and/or light chain constant region is a mouse constant region. In one embodiment, the non-human heavy and/or light chain constant region is a rat constant region. In one embodiment, the non-human heavy and/or light chain constant region is a hamster constant region.

In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof is ectopic in cells and/or tissues. In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof is at the same position compared to the wild-type non-human ADAM6 site. In one embodiment, the non-human cell and/or tissue is derived from a mouse, and the nucleotide sequence encodes a mouse ADAM6 protein or a functional fragment thereof, and is present at an ectopic location. In one embodiment, the non-human cell and/or tissue is derived from a mouse, and the nucleotide sequence encodes a mouse ADAM6 protein or a functional fragment thereof, and is present in an immunoglobulin gene segment. In a specific embodiment, the immunoglobulin gene segment is a heavy chain gene segment. In one embodiment, the contiguous sequence of the endogenous heavy chain gene segment is ectopically located in a non-human animal, wherein the contiguous sequence ectopically located in the endogenous heavy chain gene segment is a functional ADAM6 gene in a mouse (e.g., in a male mouse). Includes.

In one aspect, the use of a non-human animal as described herein to make an antigen-binding protein is provided, wherein the non-human animal comprises (a) (i) a human Vλ domain and a non-human light chain constant region. An antibody comprising an immunoglobulin light chain comprising and (ii) _{an immunoglobulin heavy chain comprising a human V H} domain and a non-human constant region; And (b) expressing the ADAM6 protein or a functional fragment thereof. In one embodiment, the antigen binding protein is human. In one embodiment, the non-human animal is a rodent and the non-human constant region is a rodent constant region. In a specific embodiment, the rodent is a mouse.

In one aspect, a non-human cell or tissue derived from a non-human animal as described herein is provided. In one embodiment, the non-human cell or tissue comprises a non-human immunoglobulin light chain constant region gene and one or more human immunoglobulin Vλ gene segments and at least one human immunoglobulin Jλ gene segment and a non-human immunoglobulin heavy chain. _{One or more human V H} , one or more human D _H and one or more human J _H gene segments contiguous with the constant region gene, wherein the cell or tissue expresses the ADAM6 protein or a functional fragment thereof. In one embodiment, the non-human light chain constant region gene is mouse Cκ or mouse Cλ.

In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof is ectopic. In one embodiment, the nucleotide sequence encoding the ADAM6 protein or functional fragment thereof is located at the same location as the wild-type non-human cell. In various embodiments, the non-human cell is a mouse B cell. In various embodiments, the non-human cell is an embryonic stem cell.

In one embodiment, the tissue is derived from the spleen, bone marrow or lymph nodes of a non-human animal.

In one aspect, the use of cells or tissues derived from a non-human animal as described herein is provided to make a hybridoma or quadroma.

In one aspect, a non-human cell comprising a modified genome as described herein is provided, wherein the non-human cell is an oocyte, a host embryo, or a cell from a non-human animal as described herein. And fusions of cells from different non-human animals.

In one aspect, the use of cells or tissues derived from non-human animals as described herein is provided to make fully human antibodies. In one embodiment, the fully human antibody comprises a human V _H domain and a human Vλ domain isolated from a non-human animal as described herein.

In one aspect, a method for making an antibody bound to an antigen of interest is provided, the method comprising (a) exposing a non-human animal as described herein to an antigen of interest, (b) a non- Isolating one or more B lymphocytes of a human animal, wherein the one or more B lymphocytes express an antibody that binds to the antigen of interest, and (c) a nucleic acid sequence encoding the immunoglobulin light chain of the antibody bound to the antigen of interest. Wherein the immunoglobulin light chain comprises a human Vλ domain and a non-human light chain constant domain, and (d) having a human immunoglobulin light chain constant region nucleic acid sequence to bind a human antibody to the antigen of interest (c) And using the nucleic acid sequence of.

In one embodiment, the non-human light chain constant domain is a mouse CK. In one embodiment, the non-human light chain constant domain is a mouse Cλ. In one embodiment, the non-human animal is a mouse.

In one aspect, a male fertile mouse comprising a modification at the immunoglobulin heavy chain site is provided, wherein the male fertile mouse comprises an ectopic ADAM6 sequence that is functional in the male mouse.

Ectopic ADAM6 in humanized heavy chain mice

Occurs in gene targeting, for example, occurrence of a bacterial artificial chromosome (BAC) now makes it possible to relatively massive recombination of genomic fragments. BAC genetic engineering allowed large deletions, and the ability to make large insertions into mouse ES cells.

Mice making human antibodies are now available for some time. While they have shown significant advances in the development of human therapeutic antibodies, these mice display a number of significant abnormalities that limit their usefulness. For example, they indicate weakened B cell development. Attenuated development may be due to various differences between transgenic and wild-type mice.

Human antibodies may not interact optimally with mouse pre B cells or B cells on the surface of mouse cells that signal for maturation, proliferation or survival during clonal selection. Fully human antibodies may not interact optimally with the mouse Fc receptor system; Mice express an Fc receptor that does not show a one-to-one correspondence with a human Fc receptor. Finally, the various mouse to create a complete human antibody does not include all the real mouse position control sequences, such as other components that may be necessary, for example, the downstream enhancer element and a wild type B cells occurs.

Mice making fully human antibodies generally contain endogenous immunoglobulin sites that are disabled in some way, and human transgenes comprising variable and constant immunoglobulin gene segments are introduced into random locations in the mouse genome. If the endogenous site is not sufficiently disabled so as not to rearrange the gene segments that form the functional immunoglobulin gene, the goal of making fully human antibodies in these mice, albeit with attenuated B cell development, can be achieved.

While making fully human antibodies from human transgenic loci is attractive, making human antibodies in mice is an obviously unfavorable process. In some mice, the process is unfavorable with respect to leading to the formation of chimeric human variable/mouse constant heavy chains (but not light chains) through a mechanism of trans-transformation. By this mechanism, a transcript encoding a fully human antibody undergoes an isoform that converts from a human isotype to a mouse isotype into trans.

The process is trans, since the fully human transgene is located away from the endogenous site that holds an intact copy of the mouse heavy chain constant region gene. Trans conversion in these mice is readily apparent, but the phenomenon is still insufficient to rescue B cell development, which remains virtually conferred. In any event, trans-converted antibodies made in these mice retain the intact human light chain, but the phenomenon of trans-conversion does not occur with respect to the light chain; This is because trans-conversion is putatively dependent on the conversion sequence in the endogenous site used (albeit different) in the normal isotype conversion in cis. Thus, even when a mouse genetically engineered to make a fully human antibody chooses a trans conversion mechanism to make an antibody with a mouse constant region, the strategy is insufficient to rescue normal B cell development. The main problem in making antibody-based human therapeutics is human immunoglobulin variable region sequences to identify useful variable domains that specifically recognize specific epitopes and bind them with a desired affinity-usually-but not always-with high affinity. Is to create a sufficiently large variety of. Before the development of VELOCIMMUNE (registered trademark) mice (described herein), mice expressing a human variable region together with a mouse constant region show some significant difference from a mouse made a human antibody from a transgene. There were no signs. However, the estimate is not accurate.

VELOCIMMUNE® mice containing the correct replacement of the mouse immunoglobulin variable region together with the human immunoglobulin variable region in the endogenous mouse locus show surprising and marked similarities to wild-type mice for B cell development. In a surprising and unexpected occurrence, VELOCIMMUNE® mice exhibited an essentially normal, wild-type response to different immunizations in only the variable region-one significant aspect from wild-type mice made for immunization.

Velocimun (VELOCIMMUNE) mice are the exact, large-scale replacement of the germline variable region with the corresponding human immunoglobulin variable region at the endogenous site of the mouse immunoglobulin heavy chain (IgH) and the immunoglobulin light chain (e.g., κ light chain, Igκ). Contains. In total, about 6 megabases of the mouse locus are replaced by about 1.5 megamaces of the human genomic sequence. This exact replacement results in mice with hybrid immunoglobulin sites that make up human variable regions and mouse constant regions heavy and light chains. Correct replacement of the mouse V _H -D _H -J _H and Vκ-Jκ segments leaves flanking mouse sequence integrity and functionality at the hybrid immunoglobulin site. The humoral immune system of mice functions like that of wild-type mice. B cell development is not steric hindrance in any significant subject and the rich diversity of human variable regions is created in mice upon antigen challenge.

VELOCIMMUNE® mice are possible because the immunoglobulin gene segments for the heavy and κ light chains are similarly rearranged in humans and mice, although they are not identical or even nearly identical (not clearly so). . However, the locus contains approximately 3 million base pairs of contiguous mouse sequences containing _{all V H} , D _H , and J _{H gene segments.} It is sufficiently similar that it can be done by replacing it with about a million bases in the genomic sequence.

In certain embodiments, further replacement of a specific mouse constant region gene sequence with a human gene sequence (e.g., replacing a mouse C _H 1 sequence with a human C _H 1 sequence, and replacing a body mouse C _L sequence with a human C _L sequence. ) Results in mice with hybrid immunoglobulin sites, making antibodies with, for example, fully human antibody fragments, such as human variable regions and partial human constant regions suitable for making fully human Fabs. Mice with hybrid immunoglobulin sites exhibit normal variable gene segment rearrangements, normal somatic hypermutations, and normal sorting shifts. These mice display a humoral immune system indistinguishable from wild-type mice, and show normal cell populations at all stages of B cell development and rescue of normal lymphoid organs, even when mice do not have a full repertoire of human variable region gene segments. Immunizing these mice results in a strong humoral response indicating the use of a wide variety of variable gene segments.

The correct replacement of the mouse germline variable region gene segment allows the mouse to partially have a human immunoglobulin site. Partly because human immunoglobulin sites are rearranged, hypermutated, and normally class converted, partly human immunoglobulin sites make antibodies in mice containing human variable regions. The nucleotide sequence encoding the variable region can be identified, cloned, and then fused with any selection sequence, such as any immunoglobulin isotype suitable for a particular use (e.g., in an in vitro system), which It results in an antibody or antigen-binding protein derived entirely from human sequences.

Large-scale humanization by recombination methods was performed using up to three megabases of mouse heavy chain immunoglobulin loci, including all _{of the mouse V H} , D _H , and J _{H gene segments, of all human V H} , D _H , and J _H gene segments. It has been used to modify mouse embryonic stem (ES) cells to accurately replace equivalent human gene segments with one megabase human genomic sequence containing some or essentially all of them. Mouse immunoglobulin κ light chain sites containing essentially all mouse Vκ and Jκ gene segments up to half of the megabase segment of the human genome, including one of two repeats encoding essentially all human Vκ and Jκ gene segments Was used to replace the three megabass segments of the.

Mice with these replaced immunoglobulin sites may contain disruption or deletion of the endogenous mouse ADAM6 site, which is between the 3′ most V _H gene segments and the 5′ most D _H gene segments at the mouse immunoglobulin heavy chain site. Found normally. Destruction in this region can result in a reduction or elimination of the functionality of the endogenous mouse ADAM6 site. If the 3′ majority of the V _H gene segments of the human heavy chain repertoire are used in the replacement, the intergenic region containing the pseudogene that has been shown to be a human ADAM6 pseudogene is between these V _H gene segments, i.e. human V _H 1-2 And V _H exist between 1-6. However, male mice containing this human intergenic sequence show a decrease in fertility. Mice are described that comprise the replaced site as described above, and that also comprise an ectopic nucleic acid sequence encoding ADAM6, wherein the mice exhibit essentially normal fertility. In one embodiment, the ectopic nucleic acid sequence comprises a mouse ADAM6a and/or mouse ADAM6b sequence or a functional fragment thereof located between the _{human V H} 1-2 gene segment and the human V _{H 6-1 gene segment at the modified endogenous heavy chain locus.} Includes. In one embodiment, the ectopic nucleic acid sequence is SEQ ID NO: 3 located between _{human V H} 1-2 and V _{H 1-6 at the modified endogenous heavy chain site.} The transcription direction of the ADAM6 gene of SEQ ID NO: 3 is opposite to that of the surrounding human V _H gene segment. The examples herein _{show the rescue of fertility by placing ectopic sequences between the indicated human V H} gene segments, but those of skill in the art will be able to determine the placement of the ectopic sequence at any suitable transcriptionally acceptable site (or even out of the chromosome) in the mouse genome. It will be appreciated that it is expected to similarly rescue fertility in male mice.

The phenomenon of replenishing mice without a functional ADAM6 site having an ectopic sequence comprising a mouse ADAM6 gene or an ortholog or homolog or a functional fragment thereof is to rescue any mouse with a non-functional or minimally functional endogenous ADAM6 site. This is the general method applicable. Thus, a large number of mice containing ADAM6-destructive modifications of the immunoglobulin heavy chain locus can be rescued by the compositions and methods of the present invention. Thus, the present invention includes mice with a wide variety of immunoglobulin heavy chain sites that attenuate endogenous ADAM6 function. Some (non-limiting) examples are provided in this description. Additionally, VELOCIMMUNE® mice are described, and the compositions and methods associated with ADAM6 can be used in a very large number of applications, such as when modifying the heavy chain locus in a wide variety of methods.

In one aspect, functional ADAM6 protein (or ortholog or homolog or functional fragment thereof), all or substantially all mouse V _H gene segments replaced with one or more human V _H gene segments, all or substantially all mouse D _H A mouse comprising a gene segment and an ectopic ADAM6 sequence encoding a replacement of the J _H gene segment with a human D _H and human J _{H gene segment is provided;} Mice do not have C _H 1 and/or hinge regions. In one embodiment, the mouse comprises: (a) human V _H -mouse C _H 1-mouse C _H 2-mouse C _H 3; (b) human V _H -mouse hinge-mouse C _H 2-mouse C _H 3; And, (c) a single variable domain binding protein that is a dimer of an immunoglobulin chain selected from _{human V H} -mouse C _H 2-mouse C _{H 3.}

In one aspect, the nucleotide sequence that rescues fertility is one or more human immunoglobulin heavy chain variable gene segments (hV _{H ) of one or more mouse immunoglobulin heavy chain variable gene segments (segments of mV H} , mD _H , and/or mJ _H). , hD _H , and/or _{segments of hJ H} ) are located within the human immunoglobulin heavy chain variable region sequence (e.g. , between the human V _H 1-2 and V _{H 1-6 gene segments) in mice having a replacement, and the mouse is} One or more mouse immunoglobulin κ light chain variable gene segments (segments of mVκ and/or mJκ) with one or more human immunoglobulin κ light chain variable gene segments (segments of hVκ and/or hJκ). In one embodiment, the nucleotide sequence is _{located between the human V H} 1-2 gene segment and the human V _H 1-6 gene segment in a Velocimmune® mouse (United States of America, incorporated herein by reference). 6,596,541 and US 7,105,348). In one embodiment, the VELOCIMMUNE® mouse thus modified is all or substantially all human immunoglobulin heavy chain variable gene segments (all hV _H , hD _H , and hJ _H segments) and all or substantially All human immunoglobulin κ light chain variable gene segments (segments of hVκ and hJκ) include choche.

In one embodiment, the one or more mouse immunoglobulin heavy chain variable gene segments comprise about 3 megabases of a mouse immunoglobulin heavy chain site. In one embodiment, the one or more mouse immunoglobulin heavy chain variable gene segments comprise at least 89 V _H gene segments, at least 13 D _H gene segments, at least 4 J _H gene segments, or a combination thereof of the mouse immunoglobulin heavy chain locus. Includes. In one embodiment, the one or more human immunoglobulin heavy chain variable gene segments comprise about 1 megabase of human immunoglobulin heavy chain sites. In one embodiment, the one or more human immunoglobulin heavy chain variable gene segments comprise at least 80 V _H gene segments, at least 27 D _H gene segments, at least 6 J _H gene segments, or combinations thereof of the human immunoglobulin heavy chain locus. Includes.

In one embodiment, the one or more mouse immunoglobulin κ light chain variable gene segments comprise about 3 megabases of a mouse immunoglobulin κ light chain site. In one embodiment, the one or more mouse immunoglobulin κ light chain variable gene segments comprise at least 137 Vκ gene segments, at least 5 Jκ gene segments, or combinations thereof of a mouse immunoglobulin κ light chain site. In one embodiment, the at least one human immunoglobulin κ light chain variable gene segment comprises about 1/2 megabase of a human immunoglobulin κ light chain site. In a specific embodiment, the at least one human immunoglobulin κ light chain variable gene segment comprises a proximal repeat of a human immunoglobulin κ light chain site (relative to the immunoglobulin κ constant region). In one embodiment, the one or more human immunoglobulin κ light chain variable gene segments comprise at least 40Vκ gene segments, at least 5 Jκ gene segments, or a combination thereof of a human immunoglobulin κ light chain site.

In one embodiment, the nucleotide sequence is located between two human immunoglobulin gene segments. In a specific embodiment, the two human immunoglobulin gene segments are heavy chain gene segments.

In one aspect, the functional mouse ADAM6 site (or ortholog or homolog or functional fragment thereof) is present in the middle of the mouse gene segment present in the endogenous mouse heavy chain variable region site, and the site contains the endogenous heavy chain constant region. It cannot be rearranged to encode a functional heavy chain. In one embodiment, the endogenous mouse heavy chain locus comprises at least 1 to 89 V _H gene segments, at least 1 to 13 D _H gene segments, at least 1 to 4 J _H gene segments, and Includes a combination of these. In various embodiments, the functional mouse ADAM6 locus (or ortholog or homolog or functional fragment thereof) encodes one or more ADAM6 proteins that are functional in the mouse, but the one or more ADAM6 proteins are SEQ ID NO: 1, SEQ ID NO: 2, and/or Includes a combination of these.

In one aspect, the functional mouse ADAM6 site (or ortholog or homolog or functional fragment thereof) is at the center of the human V _{H gene segment replacing the endogenous mouse V H gene segment.} In one embodiment, at least 89 mouse V _H gene segments are removed and _{replaced with one or more human V H} gene segments, and the mouse ADAM6 site is immediately adjacent to the _{3'end of the human V H gene segment or two human V H gene segments. It} exists between the H gene segments. In a specific embodiment, the mouse ADAM6 locus is _{between two V H} gene segments within about 20 kilobases (kb) to about 40 kilobases (kb) of the 3'end of the _{inserted human V H gene segment.} In a specific embodiment, the mouse ADAM6 site is _{between two V H} gene segments within about 29 kb to about 31 kb of the 3'end of the _{inserted human V H gene segment.} In a specific embodiment, the mouse ADAM6 site is within about 30 kb of the 3'end of the _{inserted human V H gene segment.} In a specific embodiment, the mouse ADAM6 site is within about 30,184 bp of the 3'end of the _{inserted human V H gene segment.} In a specific embodiment, the replacement comprises human V _H gene segments V _H 1-2 and V _H 6-1, and the mouse ADAM6 site is downstream of the V _H 1-2 gene segment and the V _H 6-1 gene segment Exists upstream of. In a specific embodiment, the mouse ADAM6 locus is _{present between the human V H} 1-2 gene segment and the human V _H 6-1 gene segment, but the 5'end of the mouse ADAM6 locus is 3 of the human V _H 1-2 gene segment. Is about 13,848 bp from the'end, and the 3'end of the ADAM6 site is about 29,737 bp from the 5'end of the human V _H 6-1 gene segment. In a specific embodiment, the mouse ADAM6 locus comprises SEQ ID NO: 3 or a fragment thereof that confers ADAM6 function in the cells of the mouse. In a specific embodiment, the human V _H gene segment array is the following difference in the (human V _H in the downstream from the upstream to the transcription direction of gene segments): human V _H 1-2-mouse ADAM6 spot-human V _H 6-1. In a specific embodiment, _{the ADAM6 pseudogene between human V H} 1-2 and human V _H 6-1 is replaced with a mouse ADAM6 site. In one embodiment, the orientation of at least one of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is opposite to the orientation of transcription compared to the _{orientation of the human V H gene segment.} Alternatively, the mouse ADAM6 locus resides in the intergenic region between the _{3'most human V H} gene segment and the _{5'most D H gene segment.} This can be the case if the 5'most of the D _{H segments are mouse or human.}

Similarly, mice modified with one or more human Vλ gene segments (e.g. , Vκ or Vλ segments) that replace all or substantially all endogenous mouse V _H gene segments, for example, contain a mouse ADAM6 site or a functional fragment thereof. The endogenous mouse ADAM6 site as described above is maintained by using a targeting vector with a stream homology portion, or between two human Vλ gene segments or between a human Vλ gene segment and a D _H gene segment (whether human or mouse, e.g. , Vλ + m/hD _H ), or a damaged mouse ADAM6 site with an ectopic sequence located in a J gene segment (whether human or mouse, e.g. , Vκ + J _{H ).} In one embodiment, the replacement comprises two or more human Vλ gene segments, and the mouse ADAM6 site or functional fragment thereof is present between the _{two 3′ most V L gene segments.} In a specific embodiment, the arrangement of the human Vλ gene segment is then as follows (from upstream to downstream with respect to the transcriptional direction of the human gene segment): human Vλ3' 1-mouse ADAM6 site-human Vλ3'. In one embodiment, the orientation of at least one of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus is opposite to the direction of transcription compared to the orientation of a human Vλ gene segment. Alternatively, the mouse ADAM6 locus is in the intergenic region between the _{3'most human Vλ gene segments and the 5'most D H gene segments.} This can be the case, whether the 5'most _{D H segments are mouse or human.}

In one aspect, a mouse is provided having an alteration of one or more endogenous mouse V _H gene segments and comprising at least one endogenous mouse D _{H gene segment.} In these mice, _{modifications of the endogenous mouse V H} gene segment include one or more modifications of the 3′ most V _H gene segments, but not the 5′ most D _H gene segments, where at least one 3′ most V _{Modification of the H} gene segment is processed to destroy or not create endogenous mouse ADAM6 site nonfunctionality. For example, in one embodiment the mouse comprises _{replacing all or substantially all of the endogenous mouse V H} gene segments with one or more human V _H gene segments, wherein the mouse comprises at least one endogenous D _H gene segment and a functional endogenous mouse ADAM6 Includes seats.

In another embodiment, the mouse comprises a modification of the endogenous mouse 3′ majority of the V _H gene segment, and a modification of one or more endogenous mouse D _H gene segments, wherein the modification is the endogenous mouse ADAM6 to the extent that the endogenous ADAM6 site remains functional. It is done to maintain the integrity of the seat. In one example, this modification is done in two steps: (1) 3'of the most endogenous mouse V _H gene segment using a targeting vector with an upstream homology segment and a downstream homology segment, using one or more human V _H Replacing with a gene segment, wherein the downstream homology segment comprises all or part of the functional mouse ADAM6 locus; (2) Then _{replacing the endogenous mouse D H} gene segment with a targeting vector having an upstream homology segment comprising all or a functional portion of the mouse ADAM6 site.

In various aspects, the use of mice containing an ectopic sequence encoding a mouse ADAM6 protein or an ortholog or homolog or functional homolog thereof is useful if the modification disrupts the function of the endogenous mouse ADAM6. The likelihood of disrupting endogenous mouse ADAM6 function is high when modifications are made to the mouse immunoglobulin site, especially when the mouse immunoglobulin heavy chain variable region and surrounding sequences are modified. Thus, such mice are particularly advantageous when making mice with immunoglobulin heavy chain loci that are wholly or partially deleted, wholly or partially humanized, or wholly or partially replaced (such as with VK or Vλ sequences). Methods for making the described genetic modifications for mice described below are known to those of skill in the art.

Mice containing an ectopic sequence encoding a mouse ADAM6 protein, or a substantially identical or similar protein that confers the fertility advantage of the mouse ADAM6 protein, is located at a mouse immunoglobulin heavy chain variable locus that destroys or deletes the endogenous mouse ADAM6 sequence. This is especially useful with variations on. Although described primarily with respect to mice expressing antibodies with human variable regions and mouse constant regions, these mice are useful in connection with any genetic modification that destroys the endogenous mouse ADAM6 gene. One of skill in the art will recognize that this includes a wide variety of genetically modified mice containing modifications of the mouse immunoglobulin heavy chain variable locus. These include mice that, despite other modifications, have deletions or replacements of, for example, all or part of a mouse immunoglobulin heavy chain gene segment.

Genetically modified, in certain aspects, comprising an ectopic mouse, rodent, or other ADAM6 gene (or ortholog or homolog or fragment) that is functional in a mouse, and one or more human immunoglobulin variable and/or constant region gene segments. A mouse is provided. In various embodiments, other ADAM6 gene orthologs or homologs or fragments that are functional in mice may comprise sequences from bovine, canine, primate, rabbit, or other non-human sequences.

In one aspect, replacing a mouse, all or substantially all mouse V _H gene segments encoding an ectopic ADAM6 sequence encoding a functional ADAM6 protein with one or more human V _H gene segments; Replacement of all or substantially all of the mouse D _H gene segments with one or more human D _H gene segments; And replacement of all or substantially all of the mouse J _H gene segments with one or more human J _H gene segments.

In one embodiment, the mouse further comprises a _{replacement of the mouse C H} 1 nucleotide sequence with a human C _H 1 nucleotide sequence. In one embodiment, the mouse further comprises a replacement of the mouse hinge nucleotide sequence with a human hinge nucleotide sequence. In one embodiment, the mouse further comprises a _{replacement of an immunoglobulin light chain variable site (V L} and J _L ) with a human immunoglobulin light chain variable site. In one embodiment, the mouse further comprises a replacement of a mouse immunoglobulin light chain constant region nucleotide sequence with a human immunoglobulin light chain constant region nucleotide sequence. In a specific embodiment, V _L , J _L and C _L are immunoglobulin κ light chain sequences. In a specific embodiment, the mouse comprises a mouse C _H 2 and a mouse C _{H 3 immunoglobulin constant region sequence fused to a human hinge and a human C H} 1 sequence, so that the mouse immunoglobulin site is rearranged to (a) a human variable Heavy chain with regions, human C _H 1 regions, human hinge regions, and mouse C _H 2 and mouse C _{H 3 regions;} And (b) a gene encoding an immunoglobulin light chain comprising a human variable domain and a human constant region.

In one aspect, an ectopic ADAM6 sequence encoding a functional ADAM6 protein, replacing all or substantially all mouse V _H gene segments with one or more human Vλ gene segments, and optionally all or substantially all D _H gene segments and/ Or _{replacing the J H} gene segment with one or more human D _H gene segments and/or a human J _H gene segment, or optionally replacing all or substantially all of the D _H gene segments and one or more human Jλ gene segments of the _{J H gene segment} A mouse comprising a is provided.

In one embodiment, the mouse is of all or substantially all of the mouse V _H , D _H , and J _H gene segments with one or more V _L , one or more D _H , and one or more J gene segments (e.g. , JK or J _λ ), wherein the gene segment is operably linked to the endogenous mouse hinge region, the mouse being human Vλ-human or mouse D _H -human or mouse J-mouse hinge-mouse C _H 2-mouse C _H 3 It forms a rearranged immunoglobulin chain gene containing 5'to 3'in the direction of transcription. In one embodiment, the J region is a human JK region. In one embodiment, the J region is a human J _H region. In one embodiment, the J region is a human Jλ region. In one embodiment, the human Vλ region is selected from a human Vλ region and a human Vκ region.

In a specific embodiment, the mouse is _{human Vκ, human D H} and human Jκ; Human VK, human D _H , and human J _H ; Human Vλ, human D _H , and human Jλ; Human Vλ, human D _H , and human J _H ; Human VK, human D _H , and human Jλ; A single variable domain antibody with mouse or human constant and variable regions derived from human Vλ, human D _{H, and human JK is expressed.} In a specific embodiment, the recombinant recognition sequence is modified to result in a productive rearrangement between the recited V, D and J gene segments or between the recited V and J gene segments.

In one aspect, an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or an ortholog or homolog or a functional fragment thereof), _{replacing all or substantially all of the mouse V H} gene segments with one or more human Vλ gene segments, all or substantially Provided is a mouse comprising the replacement of all mouse D _H gene segments and _{the human Jλ gene segment of the J H gene segment;} Mice do not have C _H 1 and/or hinge regions.

In one embodiment, the mouse _{lacks the sequence coding a C H} 1 domain. In one embodiment, the mouse does not have a sequence encoding the hinge region. In one embodiment, the mouse _{lacks the sequence encoding the C H} 1 domain and the hinge region.

In a specific embodiment, the mouse is thus expressing the binding protein comprising a human immunoglobulin light chain variable domain (λ, or κ) fused to the mouse C _H 2 domain, attached to the mouse C _H 3 domain.

In one aspect, an ectopic ADAM6 sequence encoding a functional ADAM6 protein (or an ortholog or homolog or a functional fragment thereof), _{replacing all or substantially all of the mouse V H} gene segments with one or more human Vλ gene segments, all or substantially As a result, mice _{encoding the replacement of all mouse D H} and J _H gene segments with human Jλ gene segments are provided.

In one embodiment, the mouse comprises a deletion of an immunoglobulin heavy chain constant region gene sequence encoding a _{C H} 1 region, a hinge region, a C _H 1 and a hinge region, or a C _H 1 region and a hinge region and a C _{H 2 region.} do.

In one embodiment, the mouse makes a single variable domain binding protein comprising a homodimer selected from: (a) human Vλ -mouse C _H 1 _{-mouse C H} 2 _{-mouse C H} 3; (b) human Vλ -mouse hinge -mouse C _H 2 _{-mouse C H} 3; (c) human Vλ-mouse C _H 2-mouse C _H 3.

In one aspect, there is provided a mouse with an endogenous heavy chain immunoglobulin site that is disabled, comprising an endogenous mouse ADAM6 site that has been disabled or deleted, wherein the mouse is a human or mouse or a nucleic acid sequence expressing a human/mouse or other chimeric antibody. Includes. In one embodiment, the nucleic acid sequence is on a transgene randomly integrated into the mouse genome. In one embodiment, the nucleic acid sequence is on an episome (eg, a chromosome) not found in wild-type mice.

In one embodiment, the mouse further comprises an endogenous immunoglobulin light chain site that has been disabled. In a specific embodiment, the endogenous immunoglobulin light chain site is selected from kappa (κ) and lambda (λ) light chain sites. In a specific embodiment, the mouse expresses an antibody comprising an endogenous κ light chain site that is disabled and a λ light chain site that is disabled, wherein the mouse expresses an antibody comprising a human immunoglobulin heavy chain variable domain and a human immunoglobulin light chain domain. In one embodiment, the human immunoglobulin light chain domain is selected from a human κ light chain domain and a human λ light chain domain. In a specific embodiment, the mouse comprises a disabled endogenous κ light chain locus, wherein the mouse comprises a human/mouse (i.e., human variable/mouse constant) immunoglobulin heavy chain and a human/mouse immunoglobulin light chain comprising a human Vλ domain. To express the antibody. In one embodiment, the human/mouse immunoglobulin light chain comprises mouse CK. In one embodiment, the human/mouse immunoglobulin light chain comprises a mouse Cλ. In a specific embodiment, mouse Cλ is Cλ2.

In one aspect, a genetically modified animal is provided that expresses a chimeric antibody and expresses an ADAM6 protein or ortholog or homolog that is functional in the genetically modified animal.

In one embodiment, the genetically modified animal is selected from mice and rats. In one embodiment, the genetically modified animal is a mouse, and the ADAM6 protein or an ortholog or homolog thereof is derived from a mouse strain that is a different strain than the genetically modified animal. In one embodiment, the genetically modified animal is a murine rodent (e.g., hamster, New World rat or mouse, ball), and the ADAM6 protein ortholog or homolog is a murine rodent (e.g., true mouse or rat, Gerbils rats, spiny pouch rats, and Japanese cypress rats). In one embodiment, the genetically modified animal is a murine rodent, and the ADAM6 protein ortholog or homolog is derived from a murine rodent.

In one embodiment, the chimeric antibody comprises a human variable domain and a rodent constant region sequence. In one embodiment, the rodent is selected from rodents of the family Rodents and rodents of the Rodentaceae family, and in a specific embodiment, the rodents of the family Rodent and the family of Rats are mice. In a specific embodiment, the rodent of the murine family and the murine family is a rat. In one embodiment, the chimeric antibody comprises a human variable domain and a constant domain from an animal selected from mouse or rat; In a specific embodiment, the mouse or rat is selected from the murine family and the murine family. In one embodiment, the chimeric antibody comprises a human heavy chain variable domain, human light chain variable domain and constant region sequence derived from a rodent selected from mouse and rat, wherein the human heavy chain variable domain and human light chain are cognate. In a specific embodiment, the cognate includes that the human heavy and human light chain variable domains are derived from a single B cell expressing the human light chain variable domain and the human heavy chain variable domain together while presenting the variable domains together on the surface of the individual B cells. do.

In one embodiment, the chimeric antibody is expressed from an immunoglobulin site. In one embodiment, the heavy chain variable domain of the chimeric antibody is expressed from a rearranged endogenous immunoglobulin heavy chain site. In one embodiment, the light chain variable domain of the chimeric antibody is expressed from a rearranged endogenous immunoglobulin light chain site. In one embodiment, the heavy chain variable domain of the chimeric antibody and/or the light chain variable domain of the chimeric antibody are expressed from a rearranged transgene (e.g., a rearranged nucleic acid sequence derived from an unrearranged nucleic acid sequence is endogenous immunity. It is integrated into the animal's genome at sites other than the globulin site). In one embodiment, the light chain variable domain of the chimeric antibody is expressed from a rearranged transgene (e.g., a rearranged sequence derived from an unrearranged nucleic acid sequence integrated into the genome of an animal at a site other than the endogenous immunoglobulin site). Nucleic acid sequence).

In a specific embodiment, the transgene is expressed from a transcriptionally active site, such as a ROSA26 site, such as a murine (eg, mouse) ROSA26 site.

In one aspect, a non-human animal is provided comprising a humanized immunoglobulin heavy chain site, wherein the humanized immunoglobulin heavy chain site comprises a non-human ADAM6 sequence or an ortholog or homolog thereof.

In one embodiment, the non-human animal is a rodent selected from mice, rats, and hamsters.

In one embodiment, the non-human ADAM6 ortholog or homolog is a sequence that is ortholog and/or homologous to the mouse ADAM6 sequence, wherein the ortholog or homolog is functional in a non-human animal.

In one embodiment, the non-human animal is selected from mice, rats, and hamsters, and the ADAM6 ortholog or homolog is derived from a non-human animal selected from mice, rats, and hamsters. In a specific embodiment, the non-human animal is a mouse and the ADAM6 ortholog or homolog is derived from an animal selected from different mouse species, rats, and hamsters. In a specific embodiment, the non-human animal is a rat and the ADAM6 ortholog or homolog is from a rodent selected from different rat species, mice, and hamsters. In a specific embodiment, the non-human animal is a hamster and the ADAM6 ortholog or homolog forms a rodent selected from different hamster species, mice, and rats.

In a specific embodiment, the non-human animal is derived from the order Muridae, and the ADAM6 sequence is derived from an animal selected from the leaping murine rodent and the murine rodent. In a specific embodiment, the rodent is a murine mouse, and the ADAM6 ortholog or homolog is derived from a murine mouse or rat or hamster.

In one embodiment, the humanized heavy chain locus comprises one or more human V _H gene segments, one or more human D _H gene segments, and one or more human J _H gene segments. In specific embodiments, one or more human V _H gene segments, one or more human D _H gene segments, and one or more human J _H gene segments are in one or more human, chimeric and/or rodent (e.g., mouse or rat) constant region genes. It is operatively connected. In one embodiment, the constant region gene is a mouse. In one embodiment, the constant region gene is a rat. In one embodiment, the constant region gene is a hamster. In one embodiment, the constant region gene comprises a _{sequence selected from hinge, C H} 2, C _H 3, and combinations thereof. In a specific embodiment, the constant region gene comprises a hinge, a C _H 2, and C _H 3 sequence.

In one embodiment, the non-human ADAM6 sequence is contiguous with a human immunoglobulin heavy chain sequence. In one embodiment, the non-human ADAM6 sequence is located within a human immunoglobulin heavy chain sequence. In a specific embodiment, the human immunoglobulin heavy chain sequence comprises V, D and/or J gene segments.

In one embodiment, the non-human ADAM6 sequence is juxtaposed with a V gene segment. In one embodiment, the non-human ADAM6 sequence is located between two V gene segments. In one embodiment, the non-human ADAM6 sequence is juxtaposed between the V and D gene segments. In one embodiment, the mouse ADAM6 sequence is located between the V and J gene segments. In one embodiment, the mouse ADAM6 sequence is juxtaposed between the D and J gene segments.

_{In one aspect, a genetically modified non-human animal comprising B cells expressing a human V H} domain cognate with a human Vλ domain from an immunoglobulin site is provided, wherein the non-human animal is a non-human animal from the immunoglobulin site. -Express immunoglobulin non-human protein. In one embodiment, the non-immunoglobulin non-human protein is an ADAM protein. In a specific embodiment, the ADAM protein is an ADAM6 protein or homolog or an ortholog or a functional fragment thereof.

In one embodiment the non-human animal is a rodent (eg, mouse or rat). In one embodiment, the rodent is murine. In one embodiment, the rodent is of the rodent family. In a specific embodiment, the rodent of the murine family is selected from mice and rats.

In one embodiment, the non-immunoglobulin non-human protein is a rodent protein. In one embodiment, the rodent is murine. In one embodiment, the rodent is of the rodent family. In a specific embodiment, the rodent is selected from mice, rats, and hamsters.

In one embodiment, the human V _H and V _L domains are attached directly or via a linker to the immunoglobulin constant domain sequence. In a specific embodiment, the constant domain sequence comprises a sequence selected from hinge, C _H 2 a C _H 3, and combinations thereof. In a specific embodiment, the human Vλ domain is selected from a Vκ or Vλ domain.

In one aspect, a genetically modified non-human animal comprising a germline human immunoglobulin sequence is provided, wherein the sperm of the male non-human animal is characterized by an in vivo migration defect. In one embodiment, the in vivo migration defect comprises the inability of a male non-human animal sperm for migration from the uterus through the fallopian tubes of a female non-human animal of the same species. In one embodiment, the non-human animal lacks the nucleotide sequence encoding the ADAM6 protein or a functional fragment thereof. In a specific embodiment, the ADAM6 protein or functional fragment thereof comprises an ADAM6a and/or ADAM6b protein or a functional fragment thereof. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from mice, rats, and hamsters.

In one aspect, a non-human animal comprising a human immunoglobulin contiguous with a non-human sequence encoding an ADAM6 protein or ortholog or homolog or functional fragment thereof is provided. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from mice, rats, and hamsters.

In one embodiment, the human immunoglobulin sequence is an immunoglobulin heavy chain sequence. In one embodiment, the immunoglobulin sequence comprises one or more V _H gene segments. In one embodiment, the human immunoglobulin sequence comprises one or more D _H gene segments. In one embodiment, the human immunoglobulin sequence comprises one or more J _H gene segments. In one embodiment, the human immunoglobulin sequence comprises one or more V _H gene segments, one or more D _H gene segments, and one or more J _H gene segments.

In one embodiment, the immunoglobulin sequence comprises one or more V _H gene segments at a high frequency in the natural human repertoire. In a specific embodiment, the at least one V _H gene segment is 2 or less V _H gene segments, 3 or less V _H gene segments, 4 or less V _H gene segments, 5 or less V _H gene segments, 6 or less V _H gene segments, 7 or less V _H gene segments, 8 or less V _H gene segments, 9 or less V _H gene segments, 10 or less V _H gene segments, 11 or less V _H gene segments, 12 or less V _H Gene segments, up to 13 V _H gene segments, up to 14 V _H gene segments, up to 15 V _H gene segments, up to 16 V _H gene segments, up to 17 V _H gene segments, up to 18 V _H gene segments , 19 or less V _H gene segments, 20 or less V _H gene segments, 21 or less V _H gene segments, 22 or less V _H gene segments, or 23 or less V _H gene segments.

In a specific embodiment, the one or more V _H gene segments comprise 5 V _H gene segments. In specific embodiments, the one or more V _H gene segments comprise 10 V _H gene segments. In a specific embodiment, the one or more V _H gene segments comprise 15 V _H gene segments. In specific embodiments, the one or more V _H gene segments comprise 20 V _H gene segments.

In various embodiments, the V _H gene segment is V _H 6-1, V _H 1-2, V _H 1-3, V _H 2-5, V _H 3-7, V _H 1-8, V _H 3 -9, V _H 3-11, V _H 3-13, V _H 3-15, V _H 3-16, V _H 1-18, V _H 3-20, V _H 3-21, V _H 3-23 , V _H 1-24, V _H 2-26, V _H 4-28, V _H 3-30, V _H 4-31, V _H 3-33, V _H 4-34, V _H 3-35, V _H 3-38, V _H 4-39, V _H 3-43, V _H 1-45, V _H 1-46, V _H 3-48, V _H 3-49, V _H 5-51, V _H 3 -53, V _H 1-58, V _H 4-59, V _H 4-61, V _H 3-64, V _H 3-66, V _H 1-69, _VH 2-70, V _H 3-72, V _H 3-73 and V _H 3-74. In various embodiments, the V _H gene segment is V _H 1-2, V _H 1-8, V _H 1-18, V _H 1-46, V _H 1-69, V _H 3-7, V _H 3 -9, V _H 3-11, V _H 3-13, V _H 3-15, V _H 3-21, V _H 3-23, V _H 3-30, V _H 3-33, V _H 3-43 , V _H 3-48, V _H 4-31, V _H 4-34, V _H 4-39, V _H 4-59, V _H 5-51 and V _H 6-1. In various embodiments, the V _H gene segment is V _H 1-18, V _H 1-46, V _H 1-69, V _H 3-7, V _H 3-11, V _H 3-15, V _H 3 -21, V _H 3-23, V _H 3-30, V _H 3-33, V _H 3-48, V _H 4-34, V _H 4-39, V _H 4-59 and V _H 5-51 Is selected from In various embodiments, the V _H gene segment is V _H 1-18, V _H 1-69, V _H 3-7, V _H 3-11, V _H 3-15, V _H 3-21, V _H 3 -23, V _H 3-30, V _H 3-43, V _H 3-48, V _H 4-39, V _H 4-59 and V _H 5-51. In various embodiments, the V _H gene segments are V _H 1-18, V _H 3-11, V _H 3-21, V _H 3-23, V _H 3-30, V _H 4-39 and V _H 4 It is selected from -59. In various embodiments, the V _H gene segment is selected from V _H 1-18, V _H 3-21, V _H 3-23, V _H 3-30 and V _H 4-39. In various embodiments, the V _H gene segment is selected from V _H 1-18, V _H 3-23 and V _H 4-39. In various embodiments, the V _H gene segment is selected from V _H 3-21, V _H 3-23 and V _H 3-30. In various embodiments, the V _H gene segment is selected from V _H 3-23, V _H 3-30 and V _H 4-39.

In a specific embodiment, the human immunoglobulin sequence comprises at least 18 V _H gene segments, 27 D _H gene segments and 6 J _H gene segments. In a specific embodiment, the human immunoglobulin sequence comprises at least 39 V _H gene segments, 27 D _H gene segments and 6 J _H gene segments. In a specific embodiment, the human immunoglobulin sequence comprises at least 80 V _H gene segments, 27 D _H gene segments and 6 J _H gene segments.

In one embodiment, the non-human animal is a mouse, and the mouse comprises _{replacement of an endogenous mouse V H} gene segment with one or more human V _H gene segments, wherein the human V _H gene segment is operable to a _{mouse C H region gene} Thus, the mouse rearranges the _{human V H} gene segment and expresses a reverse chimeric immunoglobulin heavy chain comprising a _{human V H} domain and a mouse C _H. In one embodiment, 90% to 100% of the non rearranged mouse V _H gene segment is replaced by at least one non-rearranged human V _H gene segment of. In a specific embodiment, all or substantially all of the endogenous mouse V _H gene segments are replaced with at least one unrearranged human V _H gene segment. In one embodiment, at least 19, at least 39 or at least 80 or 81 unrearranged human V _H gene segments are replaced. In one embodiment, replacement with at least 12 functional unrearranged human V _H gene segments, at least 25 functional unrearranged human V _H gene segments, or at least 43 functional unrearranged human V _{H gene segments} do. In one embodiment, the mouse comprises replacement of all mouse D _H and J _H segments with _{at least one unrearranged human D H} segment and at least one unrearranged human J _{H segment.} In one embodiment, the at least one unrearranged human D _H segment is 1-1, 1-7, 1-26, 2-8, 2-15, 3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-13, 7-27, and combinations thereof. In one embodiment, the at least one unrearranged human J _H segment is selected from 1, 2, 3, 4, 5, 6, and combinations thereof. In a specific embodiment, the one or more human V _H gene segments are 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, 6-1 Human V _H Gene segments, and combinations thereof.

In various embodiments, the human immunoglobulin sequence is operably linked with a constant region in the germline of a non-human animal (eg, a rodent such as a mouse, rat or hamster). In one embodiment, the constant region is a human, chimeric human/mouse or chimeric human/rat or chimeric human/hamster, mouse, rat, or hamster constant region. In one embodiment, the constant region is a rodent (eg, mouse or rat or hamster) constant region. In a specific embodiment, the rodent is a mouse or rat. In various embodiments, the constant region comprises at least a C _H 2 domain and a C _H 3 domain.

In one embodiment, the human immunoglobulin heavy chain sequence is located at the site of the immunoglobulin heavy chain in the germline of a non-human animal (eg, a rodent such as a mouse or rat or hamster). In one embodiment, the human immunoglobulin heavy chain sequence is located at a non-immunoglobulin heavy chain site in the germline of a non-human animal, wherein the non-heavy chain site is a transcriptionally active site. In a specific embodiment, the non-heavy chain site is the ROSA26 site.

In various aspects, the non-human animal further comprises a human immunoglobulin light chain sequence (e.g., one or more unrearranged light chain V and J sequences, or one or more rearranged VJ sequences) in the germline of the non-human animal. do. In a specific embodiment, the immunoglobulin light chain sequence is an immunoglobulin λ light chain sequence. In one embodiment, the human immunoglobulin light chain sequence comprises one or more Vλ gene segments. In one embodiment, the human immunoglobulin light chain sequence comprises one or more Jλ gene segments. In one embodiment, the human immunoglobulin light chain sequence comprises one or more Vλ gene segments and one or more Jλ gene segments.

In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 12 Vλ gene segments and one Jλ gene segment. In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 12 Vλ gene segments and 4 Jλ gene segments.

In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 28 Vλ gene segments and 1 Jλ gene segment. In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 28 Vλ gene segments and 4 Jλ gene segments.

In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 40 Vλ gene segments and 1 Jλ gene segment. In a specific embodiment, the human immunoglobulin light chain sequence comprises at least 40 Vλ gene segments and 4 Jλ gene segments.

In various embodiments, the human immunoglobulin light chain sequence is operably linked with a constant region in the germline of a non-human animal (eg, a rodent such as a mouse or rat or hamster). In one embodiment, the constant region is a human, chimeric human/rodent, mouse, rat or hamster constant region. In a specific embodiment, the constant region is a mouse or rat constant region. In a specific embodiment, the constant region is a mouse κ constant (mCκ) region or a rat κ constant (rCκ) region. In a specific embodiment, the constant region is a mouse lambda constant (mCλ) region or a rat lambda constant (rCλ) region. In one embodiment, the mouse Cλ region is a mouse Cλ2 region.

In one embodiment, the human immunoglobulin light chain sequence is located at the immunoglobulin light chain site in the germline of a non-human animal. In a specific embodiment, the immunoglobulin light chain site in the germline of the non-human animal is an immunoglobulin κ light chain site. In a specific embodiment, the immunoglobulin light chain site in the germline of the non-human animal is an immunoglobulin λ light chain site. In one embodiment, the human immunoglobulin light chain sequence is located at a non-immunoglobulin light chain site in the germline of a transcriptionally active non-human animal. In a specific embodiment, the non-immunoglobulin site is the ROSA26 site.

In one aspect, a method of making a human antibody is provided, wherein the human antibody comprises a variable domain derived from one or more variable region nucleic acid sequences encoded in a cell of a non-human animal as described herein.

In one aspect, a pharmaceutical composition comprising a polypeptide comprising an antibody or antibody fragment derived from one or more variable region nucleic acid sequences isolated from a non-human animal as described herein is provided. In one embodiment, the polypeptide is an antibody. In one embodiment, the polypeptide is a heavy chain only antibody. In one embodiment, the polypeptide is a single chain variable fragment (eg, scFv).

In one aspect, the use of a non-human animal as described herein to make an antibody is provided. In various embodiments, the antibody comprises one or more variable domains derived from one or more variable region nucleic acid sequences isolated from a non-human animal. In a specific embodiment, the variable region nucleic acid sequence comprises an immunoglobulin heavy chain gene segment. In a specific embodiment, the variable region nucleic acid sequence comprises an immunoglobulin light chain gene segment.

Mice expressing human λ variable domain

Genetically modified non-human animals (e.g., mice, rats, etc.) comprising modifications that reduce fertility due to loss of ADAM protein activity (e.g., ADAM6-dependent) are endogenous non-humans, or (e.g., gene Transplantation) human, non-human animals as described herein comprising a human λ variable sequence in a constant light chain gene. For example, non-human animals, such as mice or rats, including animals with damaged ADAM6 genes (or deleted ADAM6 genes), such as humanized immunoglobulin heavy chain loci, are human or non-human (e.g., endogenous mice or rats). ) In combination with a mouse comprising a human λ segment linked to a light chain constant region gene and a light chain locus (endogenous or transgenic) comprising a Jλ segment, but non-human animals contain the activity of recovering ADAM-dependent fertility. ADAM- modified to restore the fertility-dependent property is a non-human animal may be in at, for example, a mouse having a humanized heavy chain, or a humanized mouse having λ variable segment. The offspring form a humanized heavy chain (i.e., resulting in expressing a human heavy chain variable domain) and a humanized light chain locus (i.e., resulting in expressing a human λ light chain variable domain fused to a human or non-human λ or κ region). Including genes, but animals exhibit increased fertility compared to mice without ADAM6 activity or the activity of an ortholog or homolog of ADAM6.

VELOCIMMUNE(R) genetically engineered mice contain replacement of the unrearranged V(D)J gene segment in the endogenous mouse locus carrying the human V(D)J gene segment. VELOCIMMUNE® mice express chimeric antibodies with human variable domains and mouse constant domains (see, eg , US Pat. No. 7,605,237). Most other reports relate to mice expressing intact human antibodies from intact human transgenes in mice with incapacitated endogenous immunoglobulin sites.

The antibody light chain is encoded by one of two distinct sites: kappa (κ) and lambda (λ). The mouse antibody light chain is predominantly κ type. Mice making mouse antibodies, and modified mice making fully human or chimeric human-mouse antibodies, show bias in light chain use. Humans also exhibit light chain bias, but not as pronounced as in mice; The ratio of κ light chain to λ light chain in mice is about 95:5, whereas in humans the ratio is about 60:40. The more pronounced bias in mice is not thought to seriously affect antibody diversity, as the λ variable site in mice is not so diverse in the first example. This is not the case in humans. The human λ light chain site is richly diverse.

The human λ light chain locus extends beyond 1,000 kb and contains more than 80 genes encoding variable (V) or binding (J) segments (FIG. 19 ). Within the human λ light chain locus, more than half of all observed Vλ domains are encoded by gene segments 1-40, 1-44, 2-8, 2-14 and 3-21. Overall, it is believed that about 30 or so of the human Vλ gene segments are functional. There are 7 Jλ gene segments, of which only 4 are generally considered functional Jλ gene segments Jλ1, Jλ2, Jλ3 and Jλ7.

In humans, the λ light chain site is similar in structure to the λ site in both mouse and human, ie, the human λ light chain site has several variable region gene segments that can be recombined to form a functional light chain protein. The human λ light chain locus contains approximately 70 V gene segments and 7 Jλ-Cλ gene segment pairs. Only four of these Jλ-Cλ gene segment pairs appear to be functional. In some alleles, the fifth Jλ-Cλ gene segment pair is known to be a pseudogene (Cλ6). The 70 Vλ gene segment appears to contain 38 functional gene segments. The 70 Vλ sequences are arranged in three clusters, all of which contain different members of distinct V gene family groups (Clusters A, B and C; Figure 19). It is a potentially abundant source of relatively unused diversity for making antibodies with human V regions in non-human animals.

In stark contrast, the mouse λ light chain locus contains only 2 or 3 (depending on strain) mouse Vλ region gene segments (Figure 20). For this reason, at least, severe κ-bias in mice are not considered particularly detrimental to the overall antibody diversity.

According to the published map of the mouse λ light chain locus, the locus consists essentially of two clusters of gene segments within a range of approximately 200 kb (FIG. 20 ). The two clusters contain two sets of V, J and C genes that enable independent rearrangements Vλ2-Jλ2-Cλ2-Jλ4-Cλ4 and Vλ1-Jλ3-Cλ3-Jλ1-Cλ1. Vλ2 was found to recombine with all Jλ gene segments, and Vλ1 was found to recombine exclusively with Cλ1. Cλ4 is believed to be a pseudogene with a defective splice site.

The mouse κ light chain sites are markedly different. The structure and number of gene segments that participate in recombination events resulting in functional light chain proteins from the mouse κ site are much more complex (FIG. 21 ). Thus, the mouse λ light chain does not significantly contribute to the diversity of the antibody population in a typical mouse.

Utilizing the rich diversity of human λ light chain loci in mice is likely to result in a source for a more complete human repertoire, particularly the light chain V domain. Previous attempts to exploit this diversity have used human transgenes containing chunks of human λ light chain sites randomly incorporated into the mouse genome (e.g. , U.S. Patent No. 6,998,514 and U.S. Patent No. 7,435,871). . Mice containing these randomly integrated transgenes are known to express the intact human λ light chain, but in some cases, however, one or both of the endogenous light chain sites remain intact. This situation is undesirable as a human λ light chain sequence that competes with the mouse light chain (κ or λ) in the expressed antibody repertoire of mice.

In contrast, the present inventors describe genetically modified mice capable of expressing one or more λ light chain nucleic acid sequences directly from the mouse light chain site involved by replacement at the endogenous mouse light chain site. Genetically modified mice capable of expressing a human λ light chain sequence from an endogenous locus can be further bred to mice comprising a human heavy chain locus, thus expressing an antibody comprising the fully human V region (medium and light). It is used to make. In various embodiments. The V region is expressed together with the mouse constant region. In various embodiments, the endogenous mouse immunoglobulin gene segment is absent and the V region is expressed together with the human constant region. These antibodies have proven useful diagnostically as well as therapeutically in a number of applications.

A number of advantages can be realized for various embodiments expressing binding proteins derived from human Vλ and Jλ gene segments in mice. Advantages can be realized by placing the human λ sequence at an endogenous light chain site, eg, a mouse κ or λ site. Antibodies produced from these mice mouse C _L region, and specifically can have a light chain comprising a human Vλ domain fused to a mouse Cκ or Cλ region. Mice will also _{express a human C L} region, specifically a human Vλ domain suitable for identification and cloning for use with a CK and/or Cλ region. Since B cell development in these mice is otherwise normal, it is possible to create a Vλ domain (including a somatic mutated Vλ domain) that is compatible in the content of the Cλ or Cκ region. A genetically modified mouse comprising a Vλ gene segment that is not rearranged at the immunoglobulin κ or λ light chain site is described. Mice expressing an antibody comprising a light chain having a human Vλ domain fused to a Cκ and/or Cλ region are described.

In one aspect, (1) at least one unrearranged human Vλ gene segment and at least one unrearranged human Jλ gene segment at the endogenous immunoglobulin light chain site of a non-human animal, (2) endogenous in a non-human animal _{Genetically modified non-human animals comprising at least one human V H} gene segment, at least one human D _H gene segment, and at least one human J _H gene segment at the immunoglobulin heavy chain site are described, wherein the non-human animal is the ADAM6 protein. Or a functional fragment thereof, and the ADAM6 protein is functional in males of non-human animals. In one aspect, _{a genetically modified non-human animal expressing an antibody containing a heavy chain comprising a human V H} domain and a non-human heavy chain constant region and a light chain comprising a human Vλ domain and a non-human light chain constant region is As described, non-human animals are capable of expressing the ADAM6 protein or a functional fragment thereof. In various embodiments, the non-human animal is a rodent. In one embodiment, the rodent is a mouse or rat.

In one embodiment, the non-human light chain constant domain is a Cκ or Cλ domain. In one embodiment, the ADAM6 protein or functional fragment thereof is encoded by an ectopic sequence in the germline of a mouse. In one embodiment, the ADAM6 protein or functional fragment thereof is encoded by the endogenous sequence of a non-human animal.

In one embodiment, the endogenous light chain site of the non-human animal is an immunoglobulin λ light chain site. In one embodiment, the endogenous light chain site of the non-human animal is an immunoglobulin κ light chain site.

In one embodiment, the non-human animal _{lacks endogenous V L} and/or Jλ gene segments at the endogenous light chain locus. In a specific embodiment, the V _L and/or Jλ gene segment is a Vκ and/or Jκ gene segment. In a specific embodiment, the VL and/or Jλ gene segment is a Vλ and/or Jλ gene segment.

In one embodiment, the V _L and Jλ gene segments of a non-human animal are replaced by one or more human Vλ and one or more human Jλ gene segments. In a specific embodiment, the V _L and Jλ gene segments of the non-human animal are κ gene segments. In a specific embodiment, the V _L and Jλ gene segments of the non-human animal are λ gene segments.

In one embodiment, the one or more human Vλ gene segments are derived from a fragment of cluster A of the human immunoglobulin λ light chain locus. In a specific embodiment, a fragment of cluster A extends from human Vλ3-27 through human Vλ3-1. In a specific embodiment, a fragment of cluster A extends from human Vλ3-12 through human Jλ1. In one embodiment, the one or more human Vλ gene segments are derived from a fragment of cluster B of the human immunoglobulin λ light chain locus. In a specific embodiment, the fragment of cluster B extends from human Vλ5-52 through human Vλ1-40. In a specific embodiment, the one or more human Vλ gene segments are derived from a fragment of cluster A and a fragment of cluster B of a human immunoglobulin λ light chain site as described herein.

In one embodiment, the non-human animal comprises at least 12 human Vλ gene segments. In one embodiment, the non-human animal comprises at least 28 human Vλ gene segments. In one embodiment, the non-human animal comprises at least 40 human Vλ gene segments.

In one embodiment, the at least one human Jλ gene segment is selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof.

In one aspect, a fertile non-human male animal is provided, wherein the fertile non-human animal comprises (1) an immunoglobulin light chain comprising a human Vλ domain or a human Vκ domain, and (2) an immunity comprising a _{human V H domain.} A globulin heavy chain, and male non-human animals contain a modified heavy chain variable region site and an ectopic ADAM6 gene that is functional in male non-human animals. In one embodiment, the male non-human animal is a mouse.

In one aspect, the use of a non-human animal as described herein to make an antigen-binding protein is provided. In one embodiment, the antigen-binding protein is human. In one embodiment, the antigen-binding protein is an antibody. In one embodiment, the antigen-binding protein comprises a human V _H domain and/or a human Vλ domain derived from a non-human animal as described herein.

In one aspect, cells or tissues derived from a non-human animal as described herein are provided. In one embodiment, the tissue is derived from the spleen, bone marrow or lymph nodes. In one embodiment, the cell is a B cell. In one embodiment, the cell is an embryonic stem (ES) cell. In one embodiment, the cell is a germ cell.

In one aspect, an oocyte comprising the diploid genome of a genetically modified non-human animal as described herein is provided.

Immunoglobulin κ light chain site infertile transcript

Modifications to subjects that express human immunoglobulin λ sequences in mice are reflected in various embodiments of genetically modified mice capable of such expression. Thus, in certain embodiments, the genetically modified mouse contains specific non-coding sequence(s) from the human locus. In one embodiment, the genetically modified mouse comprises human Vλ and Jλ gene segments at the endogenous κ light chain locus, and further comprises a human κ light chain genomic fragment. In a specific embodiment, the human κ light chain genomic fragment is a non-coding sequence naturally found between a human Vκ gene segment and a human Jκ gene segment.

The human and mouse κ light chain loci encode infertile transcripts without either a start codon or an open reading frame, and contain sequences that are considered to be components that regulate transcription of the κ light chain locus. These infertile transcripts arise from intergenic sequences located downstream of most proximal Vκ gene segments or upstream of the κ light chain intron enhancer (Eκi), which is 3'and upstream of the κ light chain constant region gene (Cκ), or 5'. . Infertile transcripts arise from rearrangements of intergenic sequences to form VκJκ1 segments fused to Cκ.

Replacing the κ light chain site upstream of the Cκ gene removes the intergenic region encoding the infertile transcript. Thus, in various embodiments, the replacement of the mouse Cκ gene with a human λ light chain gene segment upstream of the mouse κ light chain sequence contains human Vλ and Jλ gene segments, but is not a κ light chain intergenic region that does not encode infertile transcripts. Humanized mice result in the κ light chain site.

Humanization of an endogenous mouse κ light chain locus carrying a human λ light chain gene segment, as described herein, results in a significant decline in the use of the κ light chain locus associated with a significant increase in λ light chain use, wherein humanization creates an intergenic region. Remove. Thus, humanized mice without intergenic regions are useful in that they can make antibodies with human light chain variable domains (eg, human λ or κ domains), and their use from the site is reduced.

In addition, for transcription, endogenous with human Vλ and Jλ gene segments associated with the insertion of a human κ intergenic region to create a Vλ site contained between the final human Vλ gene segment and the first human Jλ gene segment, and the κ intergenic region. Humanization of the mouse κ light chain site is described; This represents a population of B cells with higher expression than sites without the κ intergenic region. This observation is consistent with the hypothesis that the intergenic region inhibits use from the endogenous λ light chain site-either directly or indirectly through a sterile transcript. Under this hypothesis, inclusion of the intergenic region results in a decrease in use at the endogenous λ light chain site and leaves mice with limited selection except for the use of a modified (λ to κ) site to make antibodies.

In various embodiments, the replacement of the mouse κ light chain sequence upstream of the mouse Cκ gene with the human λ light chain sequence is for transcription, the 3'untranslated region of the 3'most Vλ gene segment and the first human Jλ gene segment. It further comprises a human κ light chain intergenic region positioned between 5'. Alternatively, this intergenic region can be omitted from the replaced endogenous κ light chain site (upstream of the mouse Cκ gene) by making a deletion in the endogenous λ light chain site. Likewise, under this embodiment, mice make antibodies from endogenous κ light chain sites containing human λ light chain sequences.

Approaches to genetically engineer mice to express human Vλ domains

Various approaches are described for making genetically modified mice that make antibodies containing light chains with a human Vλ domain fused to an endogenous C _{L (such as CK or Cλ) region.} Genetic modifications, in various embodiments, include deletion of one or both of the endogenous light chain sites. For example, removing the mouse λ light chain from the deletion of the endogenous antibody repertoire of the first Vλ-Jλ-Cλ gene cluster and the complete or partial replacement of the Vλ-Jλ gene segment of the second gene cluster with the human Vλ-Jλ gene segment. Things can be made. Also provided are mouse embryos, cells, and targeting constructs that have been genetically modified to make mice, mouse embryos, and cells.

Deletion of one endogenous Vλ-J-Cλ gene cluster and replacement of another endogenous Vλ-Jλ-Cλ gene cluster uses relatively minimal disruption in natural antibody constant region binding and function in animals, in various embodiments, Since the endogenous Cλ gene remains intact, it therefore retains the normal functions and capabilities associated with the constant region of the endogenous heavy chain. Thus, in this embodiment, the modification does not affect other endogenous heavy chain constant regions that depend on the functional light chain constant region for assembly of a functional antibody molecule containing two heavy chains and two light chains. Additionally, in various embodiments the modification does not affect the assembly of functional membrane-bound antibody molecules involving endogenous heavy and light chains, such as the hVλ domain linked to the mouse Cλ region. Since at least one functional Cλ gene is retained at the endogenous site, animals containing replacement of the Vλ-Jλ gene segment of the endogenous Vλ-Jλ-Cλ gene cluster with the human Vλ-Jλ gene segment are present in the animal's expressed antibody repertoire. The human Vλ-Jλ gene segment must be able to create a normal λ light chain that can bind to the antigen during an immune response.

A schematic illustration (no accumulation) of the deleted endogenous mouse Vλ-Jλ-Cλ gene cluster is provided in FIG. 20. As shown, the mouse λ light chain locus is organized into two gene clusters, both of which contain functional gene segments that can be recombined to form a functional mouse λ light chain. The endogenous mouse Vλ1-Jλ3-Cλ3-Jλ1-Cλ1 gene cluster is deleted by a targeting construct (targeting vector 1) with a neomycin cassette flanking the recombination site. Another endogenous gene cluster (Vλ2-Vλ3-Jλ2-Cλ2-Jλ4-Cλ4) is partially deleted by a targeting construct (targeting vector 2) with a hygromycin-thymidine kinase cassette flanking the recombination site. In this second targeting event, the Cλ2-Jλ4-Cλ4 endogenous gene segment is retained. The second targeting construct (targeting vector 2) is constructed using a different recombination site than the recombination site in the first targeting construct (targeting vector 1), thereby selectively deleting the selection cassette after successful targeting is achieved. . The resulting double targeted site is functionally silenced, i.e., an endogenous λ light chain cannot be generated. This modified locus can be used for the insertion of human Vλ and Jλ gene segments to create an endogenous mouse λ locus containing human Vλ and Jλ gene segments, whereby upon recombination at the modified locus, the animal will have the endogenous mouse Cλ gene A λ light chain comprising rearranged human Vλ and Jλ gene segments linked to the segment is generated.

Genetically modifying a mouse to make a non-functional endogenous λ gene, in various embodiments, results in a mouse that exhibits a κ light chain exclusively in its antibody repertoire, which causes the mouse to play the role of the λ light chain in the immune response. It makes it useful for evaluation, and makes it useful for creating an antibody repertoire that includes a Vκ domain but not a Vλ domain.

Genetically modified mice expressing hV linked to the recombined mouse Cλ gene at the endogenous mouse λ light chain locus can be made by any method known in the art. A schematic illustration of the replacement of the endogenous mouse Vλ2-Vλ3-Jλ2 gene segment with the human Vλ and Jλ gene segments (no accumulation) is provided in FIG. 22A. As shown, the non-functional endogenous mouse λ light chain locus is replaced by a targeting construct (12/1-λ targeting vector) comprising a neomycin cassette flanking the recombination site. The Vλ2-Vl3-Jλ2 gene segment is replaced with a genomic fragment containing a human λ sequence comprising a 12 hVλ gene segment and a single hJλ gene segment.

Thus, this first approach locates a single hJλ gene segment and one or more hVλ gene segments at consecutive endogenous λ light chain sites (FIG. 22A ).

Further modifications to the modified endogenous λ light chain site can be achieved using similar techniques to insert more hVλ gene segments. For example, a schematic illustration of two additional targeting constructs (+16-λ and +12-λ targeting vectors) used for progressive insertion of additional human hVλ gene segments is provided in FIG. 23A. As shown, an additional genomic fragment containing a specific human hVλ gene segment is inserted into the modified endogenous λ light chain site in successive steps using the homology provided by previous insertions of the human λ light chain sequence. Upon recombination with each of the targeting constructs shown, in a sequentially low fashion, 28 additional hVλ gene segments are inserted into the modified endogenous λ light chain site. This creates a chimeric site that produces a λ light chain protein containing human Vλ and Jλ gene segments linked to the mouse Cλ gene. This approach to insert a human λ light chain gene segment at the mouse λ site retains an enhancer located downstream of the Cλ2-Jλ4-Cλ4 gene segment (labeled Enh 2.4, Enh and Enh 3.1 FIGS. 22A and 23A ). This approach results in a single modified allele at the endogenous mouse λ light chain locus (FIG. 25A ).

Compositions and methods for making mice expressing light chains comprising hVλ and Jλ gene segments operably linked to mouse Cλ gene segments, including compositions and methods for making mice expressing these genes from endogenous mouse λ light chain loci Is provided. The method comprises the steps of selectively making one endogenous mouse Vλ-Jλ-Cλ gene cluster nonfunctional (e.g. , by targeted deletion), and hVλ and Jλ gene segments at the endogenous mouse λ light chain site expressing the hVλ domain in the mouse. Including the steps of using.

Alternatively, in a second approach, the human λ light chain gene segment can be located at the endogenous κ light chain site. Genetic modification, in various embodiments, includes deletion of an endogenous κ light chain locus. For example, deletions can be made to remove the mouse κ light chain from the endogenous antibody repertoire of the mouse VK and JK gene segments. Genetically modified mouse embryos, cells, and targeting constructs for making mice, mouse embryos, and cells are also provided.

For the reasons mentioned above, deletion of mouse VK and JK gene segments uses relatively minimal disruption. A schematic depiction (no accumulation) of the deleted mouse VK and JK gene segments is provided in FIG. 21. Endogenous mouse VK and JK gene segments are deleted via recombinant enzyme-mediated deletion of mouse sequence positions between two precisely positioned targeting vectors using site-specific recombination sites, respectively. A first targeting vector (JK targeting vector) is used in a first targeting event to delete a mouse JK gene segment. A second targeting vector (Vκ targeting vector) is used in a second sequential targeting event that deletes the sequence located 5'of most distal mouse Vκ gene segments. Both targeting vectors contain site-specific recombination sites, thereby selectively deleting both and all intervening mouse κ light chain sequences of the selection cassette after successful targeting has been achieved. The resulting deleted site is functionally silenced, i.e., an endogenous κ light chain cannot be produced. This modified site can be used for the insertion of the hVλ and Jλ gene segments to create an endogenous mouse κ site containing the hVλ and Jλ gene segments, whereby upon recombination at the modified site, the animal is A λ light chain comprising the rearranged hVλ and Jλ gene segments operably linked is generated. Various targeting vectors comprising human λ light chain sequences can be used with this deleted mouse κ site to create a hybrid light chain site containing a human λ gene segment operably linked to the mouse Cκ region.

Thus, in the second approach, one or more human Vλ gene segments are located in a contiguous mouse κ light chain site with a single human Jλ gene segment (12/1-κ targeting vector, FIG. 22B).

In various embodiments, modifications to this approach can add gene segments and/or regulatory sequences to optimize the use of human λ light chain sequences from mouse κ sites within the mouse antibody repertoire.

In a third approach, one or more hVλ gene segments are located at the mouse κ light chain site contiguous with four hJλ gene sequences (12/4-κ targeting vector Figure 22B).

In a third approach, one or more hVλ gene segments are located at the mouse κ light chain site contiguous with the human κ intergenic sequence and a single hJλ gene sequence (12(κ)1-κ targeting vector, FIG. 22B).

In a fourth approach, one or more hVλ gene segments are located at the mouse κ light chain site contiguous with the human κ intergenic sequence of four hJλ gene sequences (12(κ)4-κ targeting vector Figure 22b).

All of the above approaches for inserting a human λ light chain gene segment at the mouse κ locus are the κ intron enhancer element upstream of the Cκ gene (denoted as Eκi, Figures 22B and 23B) and the 3'κ enhancer downstream of the Cκ gene ( Figures 22b and 23b) denoted as Ek33' remain. The approach results in four distinct modified alleles at the endogenous mouse κ light chain site (FIG. 25B ).

In various embodiments, the genetically modified mouse comprises a knockout of an endogenous mouse λ light chain site. In one embodiment, the λ light chain locus is knocked out by a strategy to delete the region in the range Vλ2 to Jλ2, and the region in the range Vλ1 to Cλ1 (FIG. 20 ). Any strategy that reduces or eliminates the ability of an endogenous λ light chain site to express an endogenous λ domain is suitable for use with embodiments in the present disclosure.

Lambda domain antibodies from genetically modified mice

Mice containing a human λ sequence at either the mouse κ or λ light chain locus will express a light chain comprising an hVλ region fused to a _{mouse C L (Cκ or Cλ) region.} These include (a) mice comprising a functionally silenced light chain site (eg, knockout of an endogenous mouse κ or λ light chain site); (b) a mouse comprising an endogenous mouse λ light chain locus comprising hV and hJ gene segments operably linked to an endogenous mouse Cλ gene; (c) a mouse comprising an endogenous mouse κ light chain locus comprising hVκ and hJκ gene segments operably linked to an endogenous mouse Cκ gene; (d) one κ allele includes hVκ and hJκ; The remaining κ alleles include hVλ and hJλ; One κ allele includes hVλ and hJλ, one λ allele is silenced or knocked out, or the λ allele includes both hVλ and hJλ; Two heavy chain alleles are advantageously crossed with mice, each comprising _{hV H} , hD _H , and hJ _H.

Antibodies comprising an hVλ domain expressed in relation to either Cκ or Cλ are used to make fully human antibodies by cloning a nucleic acid encoding the hVλ domain into an expression construct containing a gene encoding human Cλ. The resulting expression construct is transfected into a host cell suitable for expressing an antibody representing the complete hVλ domain fused to hCλ.

The following examples are provided for methods of making and using the methods and compositions of the present invention and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise specified, temperatures are in degrees Celsius and pressures are near atmospheric.

Example 1. Humanization of mouse immunoglobulin gene

Human and mouse bacterial artificial chromosomes (BACs) were used to engineer 13 different BAC targeting vectors (BACvec) for humanization of mouse immunoglobulin heavy and κ light chain sites. Tables 1 and 2 describe the details of the steps taken for the construction of all BACvecs used for humanization of mouse immunoglobulin heavy and κ light chain sites, respectively.

Identification of human and mouse BAC. Mouse BACs spanning the 5'and 3'ends of the immunoglobulin heavy and κ light chain sites were identified by hybridization (hybridization) of the filters spotted by the BAC library or by PCR screening the mouse BAC library DNA pool. Filters were hybridized under standard conditions using probes corresponding to the region of interest. The library pool was screened by PCR using a unique pair of primers flanking the targeted region of interest. Additional PCR using the same primers was performed to deconvolve certain wells and isolate the corresponding BAC of interest. Both BAC filters and library pools were generated from 129 SvJ mouse ES cells (Incyte Genomics/Invitrogen). Spot by BAC library (Caltech B, C, or D library and RPCI-11 library, Research Genetics/Invitrogen) by screening human BAC library pool (Caltech library, Invitrogen) by PCR-based method Human BACs covering the entire immunoglobulin heavy chain and κ light chain sites were identified by hybridization of the assigned filters or using the BAC terminal sequence database (Caltech D library, TIGR).

Construction of BACvec by bacterial homologous recombination and ligation. Bacterial homologous recombination (BHR) was performed as described (Valenzuela et al. , 2003; Zhang, Y., Buchholz, F., Muyrers, JP, and Stewart, AF (1998). A new logic for DNA engineering using recombination in Escherichia coli.Nat Genet 20, 123-128). In most cases, a PCR-induced homology box was ligated to a cloned cassette and then the ligation product was gel-isolated and electroporated into BHR competing bacteria carrying the target BAC to generate linear fragments. After selection in the appropriate antibiotic Petri dish, correctly recombined BACs were identified by PCR across both the new junctions and then subjected to restriction analysis in pulse field gels (Schwartz, DC, and Cantor, CR (1984). Separation of yeast chromosome -sized DNAs by pulsed field gradient gel electrophoresis.Cell 37, 67-75), spots were identified using primers distributed over human sequences.

_{3hV H} BACvec was constructed using three sequential BHR steps for the initial stages of humanization of the immunoglobulin heavy chain locus (Fig. 4A and Table 1). In the first step (step 1), a region homologous to the mouse immunoglobulin heavy chain locus (HB1), a gene and site-specific recombination site (e.g., loxP) that confers kanamycin resistance in bacteria and G418 resistance in animal cells (kanR). A cassette is introduced into the human parent upstream of the BAC from a human V _{H 1-3 gene segment containing.} In the second step (step 2), directly downstream from _{the final J H} segment containing a second region homologous to the mouse immunoglobulin heavy chain locus (HB2) and a gene conferring resistance to spectinomycin (specR) in bacteria. Introduce the second cassette. This second step involves deleting the human immunoglobulin heavy chain locus sequence downstream from the _{J H 6 and BAC vector chloramphenicol resistance gene (cmR).} In the third step (step 3), humans double modified using the I-CeuI site added during the first step 2 and integrated by BHR into the mouse BAC (B2) via two regions of homology (HB1 and HB2). BAC (B1) was then linearized. The drug selection for the first (cm/kan), second (spec/kan) and third (cm/kan) steps is designed to be specific to the desired product. After digestion with restriction enzymes, the modified BAC clones were analyzed by pulse field gel electrophoresis (PFGE) to determine the appropriate construction (Fig. 4b).

In a similar manner, 12 additional BACvecs were engineered to humanization of the heavy and κ light chain sites. In some cases, BAC ligation was performed instead of BHR to bind the two large BACs through the introduction of rare restriction sites into both parental BACvecs by BHR with careful placement of selectable markers. This allows for the survival of the desired ligation product upon selection by a specific drug marker combination. Recombinant BAC obtained by ligation after digestion with rare restriction enzymes was identified and screened in a manner similar to that obtained by BHR (as described above).

Transformation of embryonic stem (ES) cells and generation of mice. ES cell (F1H4) targeting was performed using the Velocigen® genetic engineering method as described (Valenzuela et al., 2003). Induction of mice from modified ES cells by blastocyst (Valenzuela et al., 2003) or 8 cell injection (Poueymirou et al., 2007) is described. Targeted ES cells and mice were identified by screening for DNA from ES cells or mice with a unique set of probes and primers in PCR-based assays (eg , FIGS. 3A, 3B and 3C). All mouse studies were supervised and approved by Regeneron's Institutional Animal Care and Use Committee (IACUC).

Karyotyping and fluorescence in situ hybridization (FISH). Karyotype analysis was performed by Coriel Cell Repositories (Coriell Institute for Medical Research, Camden, NJ). FISH was performed on targeted ES cells as described (Valenzuela et al., 2003). The probes corresponding to mouse BAC DNA or human BAC DNA were labeled by nick translation (Invitrogen) by fluorescently labeled dUTP nucleotide spectrum orange or spectrum green (Vysis).

Immunoglobulin heavy chain variable gene locus. Velocigen (registered trademark) genetic engineering technology (e.g., U.S. Patent No. 6,586,251) of about 3 million base pairs (Mb) of a contiguous mouse genomic sequence comprising all V _H , D _H and J _{H gene segments (e.g., U.S. Pat.} Valenzuela et al., 2003) of the variable region of the heavy chain site in 9 sequential steps by direct replacement of about 1 Mb of a contiguous human genomic sequence comprising the same human gene segment (Fig. 1A and Table 1). Humanization has been achieved.

The intron between the J _H gene segment and the constant region gene (JC intron) is a transcription enhancer (Neuberger, MS (1983). Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells. Embo J 2, 1373-1378), followed by an isotope. Simple repeat regions required for recombination during switching (Kataoka, T., Kawakami, T., Takahashi, N., and Honjo, T. (1980).Rearrangement of immunoglobulin gamma 1-chain gene and mechanism for heavy-chain class switch. Proc Natl Acad Sci USA 77, 919-923). The junction (proximal junction) between the _{human V H} -D _H -J _H region and the mouse C _H region is to maintain the mouse heavy chain intron enhancer and switch domains to preserve both effective expression and type switching of the humanized heavy chain locus in mice. Is selected. The exact nucleotide position of this and subsequent junctions in all replacements is possible by the use of the VELOCIGENE® genetic engineering method (see above) using bacterial homologous recombination driven by the synthesized oligonucleotide. Thus, the proximal junction is _{located about 200 bp downstream from the final J H} gene segment, and the distal junction is _{700 upstream of the V H gene segment, which is the most 5'of the human locus, and mouse V H} 1-86, also known as J558.55. It is located about 9 kb downstream from the gene segment. The mouse V _H 1-86 (J558.55) gene segment is the most distal heavy chain variable reported to be potentially active in C57BL/6 mice, although it has a poor RSS sequence in the targeted 129 alleles. It is a gene segment. The distal end of the mouse heavy chain locus may reportedly contain control elements that regulate locus expression and/or rearrangement (Pawlitzky et al., 2006).

_{Using a mouse homology section of about 75 kb, 3 V H} , all 27 D _H and 9 J _H human gene segments were inserted into the proximal end of the mouse IgH site, with a coexisting 16.6 kb deletion of the mouse genomic sequence. The first insertion of the human immunoglobulin DNA sequence into the mouse was accomplished using the 144 kb proximal end of the containing human heavy chain locus (step A, FIG. 2A; velocimun and 3, 3hV _H ). This large 144 kb insertion and concomitant 16.6 kb deletion were performed in a single step (step A) occurring with a frequency of 0.2% (Table 3). Accurately targeted ES cells were scored by natural allele loss (LONA) assay (Valenzuela et al., 2003) using probes within the deleted mouse sequence and the inserted human sequence on both sides and spanned the entire insert. Multiple probes were used to verify the integrity of the large human insert (FIGS. 3A, 3B and 3C ). Since many rounds of sequential ES cell targeting are expected, the targeted ES cell clones at this and all subsequent steps are subjected to karyotyping (see above), and these clones exhibiting normal karyotypes in at least 17 spreads out of 20. Only was used in the subsequent step.

Targeted ES cells from step A were retargeted with BACvec producing a 19 kb deletion at the distal end of the heavy chain locus (step B, Figure 2a). In contrast to the neomycin resistance gene (neo) contained in the BACvec of step A, the step B BACvec contained the hygromycin resistance gene (hyg). Upon successful targeting to the same chromosome, approximately 3 Mb of the mouse heavy chain variable locus containing all mouse V _{H gene segments other than V H} 1-86 and all D _{H gene segments other than DQ52, and 2 resistance genes.} flanked by loxP sites; Resistant genes from two BACvec are designed so that DQ52 and all mouse J _{H chain gene segments are deleted in step A.} High G418 _{driving a 3hV H} proximal cassette for homozygous (Mortensen, RM et al. (1992) Production of homozygous mutant ES cells with a single targeting construct.Mol Cell Biol 12, 2391-2395), and the fate of the distal hyg cassette. According to, double-targeted ES cell clones on the same chromosome were identified. Even in the absence of drug selection, a mouse segment of 4 Mb in size modified in such a way that it is flanked by loxP sites in ES cells by transient expression of high efficiency (less than about 11%) CRE recombinase was successfully deleted. (Zheng, B. et al. (2000) Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications.Mol Cell Biol 20, 648-655). In a similar manner, we achieved 3 Mb deletions in 8% of ES cell clones after transient CRE expression (Step C, Figure 2A; Table 3). Deletion was scored by LONA assay using probes at the ends of the deleted mouse sequence, and the presence of PCR products along the deletion point, including the loss of neo and hyg and the only remaining loxP sites. In addition, the deletion was confirmed by fluorescence in situ hybridization (data not shown).

_{The remainder of the human heavy chain variable region was added to the 3hV H} allele in a series of 5 steps using a VELOCIGENE (registered trademark) genetic manipulation method (step EH, Fig. 2b), and each step is a human of 210 kb or less. Included the correct insertion of the gene sequence. For each step, the proximal end of each new BACvec is designed to overlap with the most distal human sequence of the previous step, and the distal end of each new BACvec contains the same distal region of mouse homology used in step A. I did. The BACvec of steps D, F and H contains the neo selection cassette, and the BACvec of steps E and G contains the hyg selection cassette, so the selection is alternated between G418 and hygromycin. Targeting in step D was evaluated by the loss of unique PCR products across the distal loxP site of the 3hV _{H hybrid allele.} The targeting of steps E-I was evaluated by the loss of the previous selection cassette. In the final step (step I, Fig. 2b), a neo selection cassette with Frt sites on both sides (McLeod, M. et al. (1986) Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle Mol Cell Biol 6, 3357-3367) was removed by transient FLPe expression (Buchholz, F. et al. (1998) Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16, 657-662). The human sequences of BACvec for stages D, E and G are each derived from two parental human BACs, and those from stages F and H are derived from a single BAC. Retention of the human sequence at all steps was confirmed using multiple probes across the inserted human sequence (as described above, such as FIGS. 3A, 3B and 3C). Only these clones with normal karyotype and germline potential were carried forward at each stage. ES cells from the final stage could still contribute to the germline after 9 sequential manipulations (Table 3). Mouse homozygous for each heavy chain allele demonstrated a viable, healthy looking, and substantial wild-type humoral immune system (see Example 3).

Immunoglobulin κ light chain variable locus. Κ in 8 sequential steps by direct replacement of about 0.5 Mb of human sequence comprising proximal human Vκ and Jκ gene segments of about 3 Mb of mouse sequence comprising all Vκ and Jκ gene segments in a manner similar to the heavy chain. The light chain variable region was humanized (Figure 1B; Table 2 and Table 4).

The variable region of the human κ light chain site contains two nearly identical 400 kb repeats separated by 800 kb spacers (Weichhold, GM et al. (1993) The human immunoglobulin kappa locus consists of two copies that are organized in opposite polarity.Genomics 16 , 503-511). Since the repeats are so similar, almost all of the locus diversity can be reproduced in mice using proximal repeats. In addition, natural human alleles of the κ light chain site missing the distal repeat have been reported (Schaible, G. et al. (1993) The immunoglobulin kappa locus: polymorphism and haplotypes of Caucasoid and non-Caucasoid individuals.Hum Genet 91 , 261-267). We replaced about 3 Mb of mouse κ light chain variable gene sequence with about 0.5 Mb of human κ light chain variable gene sequence, effectively replacing all mouse Vκ and Jκ gene segments with proximal human Vκ and all human Jκ gene segments ( 2C and 2D; Tables 2 and 4). Contrary to the method described in Example 1 for the heavy chain locus, the entire mouse Vκ gene region, including all Vκ and Jκ gene segments, was deleted in a three-step process and then any human sequence was added. Initially, a neo cassette was introduced at the proximal end of the variable region (step A, Figure 2c). Next, the hyg cassette was inserted at the distal end of the κ site (step B, Fig. 2c). The LoxP sites were relocated within each selection cassette so that CRE treatment induced deletion of the remaining 3 Mb mouse Vκ regions along both resistance genes (step C, FIG. 2c).

Human genome fragments of about 480 kb in size including the entire immunoglobulin κ light chain variable region were inserted in four sequential steps (Fig. 2D; Tables 2 and 4), and humans of 150 kb or less using a method similar to that used for the heavy chain The immunoglobulin κ light chain sequence was inserted in a single step (see Example 1). The final hygromycin resistance gene was removed by transient FLPe expression. As with the heavy chain, targeted ES cell clones were evaluated for integrity, normal karyotype and germline potential of the whole human insert after each step. A mouse homozygous for each κ light chain chain allele was generated and observed to have a healthy and normal appearance.

Example 2. Generation of fully humanized mice by combination of multiple humanized immunoglobulin alleles

At several points, microinjection of ES cells bearing a portion of the human immunoglobulin heavy chain or κ light chain variable repertoire described in Example 1 and breeding the resulting mice to have a plurality of progressively larger fractions of the human germline immunoglobulin repertoire. A version of VELOCIMMUNE® mice was generated (Table 5; FIGS. 5A and 5B). VELOCIMMUNE® 1(V1) mice carry _{all human D H} and J _H gene segments and all human JK gene segments _{combined with 18 human V H} gene segments and 16 human VK gene segments. . VELOCIMMUNE® 2 (V2) and VELOCIMMUNE® (V3) mice have a total of 39 V _H and 30 Vκ, and 80 V _H and 40 Vκ, respectively. Has an increased variable repertoire. Since the genomic regions encoding the mouse V _H , D _H and J _H gene segments, and the Vκ and Jκ gene segments are completely replaced, the antibodies produced by any version of VELOCIMMUNE® mice are mouse constant. It includes a human variable region linked to the region. The mouse λ light chain locus remains non-contact in all versions of Velocimmune® mice, and acts as a comparator for the expression efficiency of the various Velocimune® κ light chain loci.

Mouse double homozygous for both immunoglobulin heavy and κ light chain humanizations was generated from a subpopulation of alleles described in Example 1. All genotypes observed during the mating process to generate double homozygous mice appeared in almost Mendelian proportions. Male offspring homozygous for each human heavy chain allele showed a decrease in fertility. The decrease in fertility resulted from loss of mouse ADAM6 activity. The mouse heavy chain variable locus contains two embedded functional ADAM6 genes (ADAM6a and ADAM6b). During humanization of the mouse heavy chain variable locus, the inserted human genomic sequence contains the ADAM6 pseudogene. Mouse ADAM6 may be required for fertility, and thus deletion of the mouse ADAM6 gene at the humanized heavy chain variable locus can reduce fertility in these mice despite the presence of a human pseudogene. Examples 7-9 again describe the correct replacement of the deleted mouse ADAM6 gene with a humanized heavy chain variable locus, and restoration of wild-type level of fertility in mice with a humanized heavy chain immunoglobulin locus.

Example 3. Lymphocyte population in mice with humanized immunoglobulin gene

The mature B cell population in three different versions of Velocimmune® mice was evaluated by flow cytometry.

1,

2,

3 경쇄-FITC, anti 마우스 Igκ 경쇄-PE, 항마우스 CD45R(B220)-APC; G: 항마우스 Ly6G/C-FITC, 항마우스 CD49b(DX5)-PE, 항마우스 CD11b-APC; H: 항마우스 CD4(L3T4)-FITC, 항마우스 CD45R(B220)-PE, 항마우스 CD8a-APC. 도 6에 결과가 도시되어 있다.Briefly, cell suspensions from bone marrow, spleen and thymus were prepared using standard methods. ^{Cells were resuspended at 5x10 5} cells/ml in BD Pharmingen FACS staining buffer, blocked with anti-mouse CD16/32 (BD Pharmingen), stained with an appropriate antibody cocktail and between BDs according to all manufacturer's instructions. It was fixed with Cytofix(trade name). The final cell pellet was resuspended in 0.5 ml staining buffer and analyzed using BD FACSCalibur™ and BD CellQuest Pro™ software. All antibodies (BD Pharmingen) were prepared in mass dilution/cocktail and added to a final concentration of ^{0.5 mg/10 5 cells.} Antibody cocktails for bone marrow (AD) staining are as follows: A: anti-mouse IgM ^b -FITC, anti-mouse IgM ^a -PE, anti-mouse CD45R(B220)-APC; B: anti-mouse CD43(S7)-PE, anti-mouse CD45R(B220)-APC; C: anti-mouse CD24 (HSA)-PE; Anti-mouse CD45R(B220)-APC; D: anti-mouse BP-1-PE, anti-mouse CD45R(B220)-APC. Antibody cocktails for spleen and inguinal lymph node (EH) staining are as follows: E: anti-mouse IgM ^b -FITC, anti-mouse IgM ^a -PE, anti-mouse CD45R(B220)-APC; F: anti-mouse Ig,

One,

2,

3 light chain-FITC, anti mouse Igκ light chain-PE, anti mouse CD45R(B220)-APC; G: anti-mouse Ly6G/C-FITC, anti-mouse CD49b (DX5)-PE, anti-mouse CD11b-APC; H: anti-mouse CD4 (L3T4)-FITC, anti-mouse CD45R (B220)-PE, anti-mouse CD8a-APC. The results are shown in Figure 6.

Lymphocytes isolated from the spleen or lymph nodes of homozygous VELOCIMMUNE® mice were stained for surface expression of markers B220 and IgM and analyzed using a flow cytometer (FIG. 6). The size of the ^{B220 +} IgM ⁺ mature B cell population in all versions of Velocimmune® mice tested were substantially the same as the wild-type mice, regardless of the number of _{V H gene segments they contained.} In addition, mice containing a homozygous hybrid humanized immunoglobulin heavy chain site, even mice with only three V _H gene segments, but a normal mouse immunoglobulin κ light chain site, or a normal mouse immunoglobulin containing a homozygous hybrid humanized κ light chain site Mice with heavy chain loci also had a ^{normal number of B220 +} IgM ⁺ cells in their peripheral compartments (not shown). These results indicate that chimeric sites with human variable gene segments and mouse constant regions are able to completely shift the mature B cell compartment. In addition, the number of variable gene segments at the heavy or κ light chain sites and thus the theoretical diversity of antibody repertoires is not correlated with the ability to generate wild-type populations of mature B cells. Conversely, mice with randomly integrated fully human immunoglobulin transgenes and inactivated mouse immunoglobulin sites have a reduced number of B cells in this compartment, and the degree of deficiency depends on the number of variable gene segments contained in the transgene (Green , LL, and Jakobovits, A. (1998).Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes. J Exp Med 188, 483-495). This indicates that the “ in situ gene humanization” strategy produces essentially different functional outcomes than the randomly integrated transgenes achieved in the “knockout-plus-transformation” approach.

Allele exclusion and site selection. The ability to maintain allele exclusion in mice that are heterozygous for different versions of humanized immunoglobulin heavy chain sites was investigated.

Humanization of immunoglobulin sites was performed in F1 ES cell lines (F1H4 (Valenzuela et al., 2003)) derived from 129S6/SvEvTac and C57BL/6NTac heterozygous embryos. The human heavy chain germline variable gene sequence ^{is targeted with the 129S6 allele carrying the IgM a} monophasic, and the unmodified mouse C576BL/6N allele carries the IgM ^b monophasic. Antibodies specific for polymorphisms found in the IgM ^a or IgM ^b alleles can be used to differentiate this allele form of IgM by flow cytometry. As shown in Figure 6 (bottom row), only B cells identified in mice that are heterozygous for each version of the humanized heavy chain locus are IgM ^a (humanized allele) or IgM ^b (wild type allele). Express the gene. This indicates that the mechanism involved in allele exclusion is intact in VELOCIMMUNE® mice. In addition, the relative number of B cells positive for the humanized allele (IgM ^a ) is almost proportional to the number of _{V H gene segments present.} The humanized immunoglobulin locus is expressed in approximately 30% of B cells in VELOCIMMUNE® 1 heterozygous mice with _{18 human V H gene segments and has 39 and 80 human V H} gene segments respectively. It was expressed in 50% of B cells in VELOCIMMUNE® 2 and 3 (not shown) heterozygous mice. In particular, the ratio of cells expressing humanized to wild-type mouse alleles (VELOCIMMUNE® 1 0.5 for mice and 0.9 for VELOCIMMUNE® 2 mice) was humanized versus wild-type sites. The ratio of the number of variable gene segments contained in (Velocimune (registered trademark) 0.2 for 1 mouse and VELOCIMMUNE (registered trademark) 2 mouse) is greater than 0.4). This may indicate that the probability of allele selection is intermediate between random selection of one or another chromosome and random selection of any particular V-segment RSS. Additionally, there may be a fraction of B cells, if not all, and one allele becomes accessible for recombination, completes this process, and stops recombination before the other allele becomes accessible. In addition, the even distribution of cells with surface IgM (sIgM) from the hybrid humanized heavy chain locus or wild-type mouse heavy chain locus is evidence that the hybrid locus works at normal levels. In contrast, randomly integrated human immunoglobulin transgenes compete poorly with wild-type mouse immunoglobulin sites (Bruggemann, M. et al. (1989) A repertoire of monoclonal antibodies with human heavy chains from transgenic mice.PNAS 86, 6709 -6713; Green et al., 1994; Tuaillon, N. et al. (1993) Human immunoglobulin heavy-chain minilocus recombination in transgenic mice: gene-segment use in mu and gamma transcripts.Proc Natl Acad Sci USA 90, 3720- 3724). This further shows that immunoglobulins produced by VELOCIMMUNE® mice are functionally different from those produced by randomly integrated transgenes in mice produced by the “knockout-plus-transformation” approach. Show.

The polymorphism of the Cκ region at 129S6 or C57BL/6N is not available to investigate the allele exclusion of the humanized versus non-humanized κ light chain site. However, VELOCIMMUNE (registered trademark) mice all possess wild-type mouse λ light chain locus, and therefore, it can be observed whether rearrangement and expression of the humanized κ light chain locus can prevent mouse λ light chain expression. The ratio of the number of cells expressing the humanized κ light chain to the number of cells expressing the mouse λ light chain was compared to the wild-type mouse, regardless of the number of human Vκ gene segments inserted at the κ light chain site. Trademark) relatively unchanged in mice (Fig. 6, the third row from the top). In addition, there is no increase in the number of double-positive (κ and λ) cells, indicating that productive recombination at the hybrid κ light chain site adequately inhibits the recombination of the mouse λ light chain site. Conversely, mice containing a randomly integrated κ light chain transgene with an inactive mouse κ light chain site-not the wild-type mouse λ light chain site-show a dramatically increased κ/λ ratio (Jakobovits, 1998), which was introduced This means that the κ light chain transgene does not work well in these mice. This further shows the different functional results observed in immunoglobulins made by VELOCIMMUNE® mice compared to immunoglobulins made by knockout-plus-transformed" mice.

B cell development. Since the mature B cell population in VELOCIMMUNE® mice resembled the population in wild-type mice (described above), defects in early B cell differentiation can be compensated for by proliferation of the mature B cell population. have. Various stages of B cell differentiation were investigated by analysis of the B cell population using a flow cytometer. Table 6 describes the proportion of the fraction of cells in each B cell lineage defined by FAC using specific cell surface markers in VELOCIMMUNE® mice compared to wild type litter.

Early B cells develop in the bone marrow, and different stages of B cell differentiation are characterized by changes in the type and amount of cell surface marker expression. This difference in surface expression correlates with molecular changes that occur at immunoglobulin sites within cells. Pro-B transition to pre-B cells requires successful rearrangement and expression of functional heavy chain proteins, and transition from pre-B to mature B stage is governed by the correct rearrangement and expression of the κ or λ light chain. Thus, an ineffective transition between stages of B cell differentiation can be detected by a change in the relative population of B cells at a given stage.

No major disadvantages of B cell differentiation were observed in any VELOCIMMUNE® mice. Introduction of the human heavy chain gene segment does not appear to affect the transition of pro-B to pre-B in VELOCIMMUNE (registered trademark) mice, and the introduction of the human κ light chain gene segment does not appear to affect the transition of pre-B to B. Does not seem to affect This indicates that "reverse chimeric" immunoglobulin molecules bearing human variable regions and mouse constant regions function normally in the context of B cell signaling and coreceptor molecules resulting in proper B cell differentiation in the mouse environment. In contrast, the balance between different populations during B cell differentiation is fluctuated to varying degrees in mice containing randomly integrated immunoglobulin transgenes and inactivated endogenous heavy or κ light chain sites (Green and Jakobovits, 1998).

Example 4. Variable gene repertoire in humanized immunoglobulin mice

Human variable genes in the humanized antibody repertoire of VELOCIMMUNE® mice by reverse transcriptase-polymerase chain reaction (RT-PCR) of human variable regions from various sources including splenocytes and hybridoma cells. The use of segments was analyzed. The variable region sequence of the rearranged variable region gene segments, the use of the gene segments, somatic hypermutation, and splicing diversity were determined.

^{Briefly, total RNA was transferred to 1x10 7} to 2x10 ⁷ splenocytes or about 10 ⁴ -10 ⁵ hybridoma cells using TRIzol® (Invitrogen) or Qiagen RNeasy® mini kit (Qiagen). Was extracted from, and primed with mouse constant region specific primers using a Superscript (trade name) III 1-step RT-PCR system (Invitrogen). Separately from each sample to 2-5 μl of RNA using the aforementioned 3'constant specific primers paired with pooled leader primers for each family of human variable regions for both heavy and κ light chains. The reaction was carried out. The volume of reagents and primers, and RT-PCR/PCR conditions were performed according to the manufacturer's instructions. Primer sequences are based on multiple sources (Wang, X. and Stollar, BD (2000) Human immunoglobulin variable region gene analysis by single cell RT-PCR. J Immunol Methods 244:217-225; Ig-primer sets, Novagen) . If appropriate, a second PCR reaction was performed fitted with the same mouse 3'immunoglobulin constant specific primer and pooled family specific framework primer used in the first reaction. Aliquots (5 μl) from each reaction were analyzed by agarose electrophoresis, and the reaction product was agarose using a Montage Gel Extraction Kit (Millipore). Purified from. The cloning of the purified product using the TOPO (TM) TA cloning system (Invitrogen) and DH10β by electroporation. E. coli cells were transformed. Each clone was selected from each transformation reaction and grown overnight at 37° C. in 2 ml LB broth culture with antibiotic selection. Plasmid DNA was purified from bacterial culture by a kit-based approach (Qiagen).

Use of immunoglobulin variable genes. Plasmid DNA of both heavy and κ light chain clones was sequenced with T7 or M13 reverse primers on an ABI 3100 Genetic Analyzer (Applied Biosystems). The sequence raw data was input into a sequencer (trade name) (v4.5, genetic code). To confirm the use of human V _H , D _H , J _H and Vκ, Jκ segments, each sequence was contiguously assembled and IMGT V-Quest (Brochet, X., Lefranc, MP, and Giudicelli, V. (2008). IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized VJ and VDJ sequence analysis.Nucleic Acids Res 36, W503-508) were aligned with human immunoglobulin sequences using the irradiation function. Sequences were compared to germline sequences for somatic hypermutation and recombination junction analysis.

Mice were subjected to initial heavy chain modification (3hV) by RAG supplementation (Chen, J. et al. (1993) RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development.Proc Natl Acad Sci USA 90, 4528-4532). _H- CRE hybrid allele, the bottom of Figure 2a) was generated from ES cells containing, and cDNA was prepared from splenocyte RNA. Amplification of cDNA using a primer set (described above) specific for the expected chimeric heavy chain mRNA resulting from V(D)J recombination within the inserted human gene segment and subsequent splicing to the mouse IgM or IgG constant domain. Made it. Sequences derived from this cDNA clone (not shown) are appropriate V(D)J recombination occurs within the human variable gene sequence, and the rearranged human V(D)J gene segment is appropriately spliced in frame to the mouse constant domain. , Indicates that class switch recombination occurs. Subsequent further sequencing of the mRNA product of the hybrid immunoglobulin site was performed.

In a similar experiment, B cells from non-immunized wild-type and VELOCIMMUNE® mice were isolated by flow cytometry based on the surface expression of B220 and IgM or IgG. B220 ⁺ IgM ⁺ IgG ⁺ or surface (sIgG ^+), pooling the cells, RT-PCR amplification and cloning (the base material) after the V _H and Vκ obtain a sequence. Recorded representative gene use in a series of RT-PCR amplified cDNAs from non-immunized VELOCIMMUNE® 1 mice (Table 7) and VELOCIMMUNE® 3 mice (Table 8). ( ^* defective RSS; loss or pseudogene).

As shown in Tables 7 and 8, almost all functional human V _H , D _H , J _H , VK and JK gene segments were used. Of the functional variable gene segments described, although not detected in the VELOCIMMUNE® mice of this experiment, some were reported to possess defective recombination signal sequences (RSS) and were not expected to be expressed accordingly (Feeney , AJ (2000) Factors that influence formation of B cell repertoire.Immunol Res 21, 195-202). Analysis of several different sets of immunoglobulin sequences from various VELOCIMMUNE® mice isolated from both intact and immunization repertoires indicated use of this gene segment despite its low frequency (data not shown). Comprehensive gene usage data show that all functional human V _H , D _H , J _H , Vκ, and Jκ gene segments contained in Velocimmune® mice were observed in various intact and immunization repertoires (data not shown). Indicates that. Although the human V _H 7-81 gene segment was identified in the analysis of the human heavy chain locus sequence (Matsuda, F. et al. (1998) The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. J Exp Med 188, 2151- 2162), as confirmed by resequencing of the whole VELOCIMMUNE® 3 mouse genome, this is not present in VELOCIMMUNE® mice.

It is known that the sequences of the heavy and light chains of antibodies exhibit unusual variability, particularly in short polypeptide segments within rearranged variable domains. This region, known as the hypervariable region or complementarity determining region (CDR), creates a binding site for the antigen in the structure of the antibody molecule. The intervening polypeptide sequence is called the framework region (FR). There are 3 CDRs (CDR1, CDR2, CDR3) and 4 FRs (FR1, FR2, FR3, FR4) in both the heavy and light chains. This CDR is unique in that one CDR, CDR3, _{is produced by recombination of both V H} , D _H and J _H and VK and JK of gene segments and produces a significant amount of repertoire diversity before hitting the antigen. This binding is inaccurate due to both nucleotide deletion through exonuclease activity and non-template encoded addition through terminal deoxynucleotide transferase (TdT), thus resulting in novel sequences from the recombination process. Although the FR as a whole may exhibit substantial somatic mutations due to the high mutagenicity of the variable region, however, the variability is not evenly distributed throughout the variable region. CDRs are variable, centralized and localized regions on the surface of antibody molecules that allow antigen binding. The heavy and light chain sequences of antibodies selected from VELOCIMMUNE® mice around the CDR3 junction showing junctional diversity are shown in Figures 7A and 7B, respectively.

As shown in Figure 7A, non-template-encoded nucleotide addition (N-addition) was observed at both the _{V H} -D _H and D _H -J _{H junctions in antibodies from VELOCIMMUNE® mice,} Shows the proper function of TdT with human segments. The _{endpoints of the V H} , D _H and J _H segments for their germline counterparts indicate that exonuclease activity also occurs. Unlike the heavy chain locus, the human κ light chain rearrangement shows little or no TdT addition in the CDR3 formed by recombination of the Vκ and Jκ segments (FIG. 7B ). This is expected as a lack of TdT expression in mice during light chain rearrangement in the transition of pre-B to B cells. The diversity observed in CDR3 of the rearranged human Vκ region is mainly introduced through exonuclease activity during recombination events.

Somatic hypermutation. It adds additional diversity to the variable regions of the immunoglobulin genes rearranged during the germinal center reaction by a process called somatic hypermutation. B cells expressing the somatic mutated variable region compete with other B cells for access to the antigen presented by the follicular dendritic cells. These B cells with higher affinity for the antigen are present peripherally after further proliferating and undergoing class switching. Thus, B cells expressing the switched isotope typically encounter antigens, undergo a germinal center reaction, and have an increased number of mutations compared to intact B cells. Additionally, variable region sequences from predominantly intact sIgM ⁺ B cells are expected to have relatively fewer mutations than variable sequences from ^{sIgG +} B cells that have undergone antigen selection.

Non-immunized Velocity myun (VELOCIMMUNE) (registered trademark) sIgM ⁺ or sIgG ⁺ B cells, or randomly from the immunized mice sIgG ⁺ B cells from the V _H or Vκ germline thereof for with germline sequence annotation sequences from the clones from the mouse Variable gene segments and changes were compared. The resulting nucleotide sequence was translated in silico and the mutation that caused the amino acid change was also annotated. Data was collected from all variable regions and the percentage change at a given point was calculated (FIG. 8 ).

As shown in Figure 8, the human heavy chain variable region derived ^{from sIgG +} B cells from non-immunized VELOCIMMUNE® mice exhibited more nucleotides compared to ^{sIgM + B cells from the same splenocyte pool,} Heavy chain variable regions from immunized mice show even more changes. The number of changes increases relative to the framework region in the complementarity determining region (CDR), indicating antigen selection. The corresponding amino acid sequence from the human heavy chain variable region also represents a significantly greater number of mutations in IgG compared to IgM, even in immunized IgG. This mutation again appears to be more common in the CDRs compared to the framework sequence, suggesting that the antibody is antigen-selected in vivo. A similar increase in the number of nucleotide and amino acid mutations was observed in the Vκ sequence from ^{IgG +} B cells from immunized mice.

The gene use and somatic hypermutation observed in VELOCIMMUNE® mice indicate that substantially all of the gene segments present can be rearranged to form a fully functional reverse chimeric antibody in these mice. In addition, the Velocimmune® antibody fully participates within the mouse immune system to undergo affinity selection and maintains the production of fully mature human antibodies that can effectively neutralize its target antigen. VELOCIMMUNE® mice are capable of initiating robust immune responses to many types of antigens using a wide range of human antibodies suitable for therapeutic use with high affinity (data not shown).

Example 5. Analysis of lymphocytic structure and serum isotope

The entire structure of the spleen, inguinal lymph nodes, Peyer patches and thymus of tissue samples from wild-type or VELOCIMMUNE® mice stained with H&E was investigated with an optical microscope. The level of immunoglobulin isotopes in serum collected from wild-type and VELOCIMMUNE® mice was analyzed using Luminex® technology.

Lymphocytic organ structure. The structure and function of lymphocytic tissue depends in part on the proper development of hematopoietic cells. Defects in B cell development or function can manifest as alterations in the structure of lymphocytic tissue. Upon analysis of the stained tissue sections, no significant difference in appearance of secondary lymphocytic organs was found between wild-type and VELOCIMMUNE® mice (data not shown).

Serum immunoglobulin levels. The expression level of each isotope is similar in wild-type and VELOCIMMUNE® mice (Fig. 9A, Fig. 9B and Fig. 9C). This indicates that humanization of variable gene segments does not have a clear adverse effect on class switching or immunoglobulin expression and secretion and thus clearly retains all endogenous mouse sequences required for this function.

Example 6. Immunization and antibody production in humanized immunoglobulin mice

Different versions of VELOCIMMUNE® mice were immunized with antigens to investigate the humoral response to foreign antigen challenge.

Immunization and hybridoma development. VELOCIMMUNE (registered trademark) and wild-type mice were immunized with antigens in the form of proteins, DNA, DNA and proteins, or in the form of cells expressing the antigen. Animals were usually boosted every 3 weeks for a total of 2 to 3 times. After each antigen boosting, serum samples from each animal were collected and analyzed for antigen specific antibody responses by serum titer determination. Prior to fusion, mice were administered 5 μg of a final pre-fusion boost of protein or DNA as desired via intraperitoneal and/or intravenous injection. Splenocytes were harvested and fused to Ag8.653 myeloma cells in an electrofusion chamber according to the manufacturer's suggested protocol (Cyto Pulse Sciences Inc., Glen Burnie, MD). After 10 days of culture, hybridomas were screened for antigen specificity using ELISA assay (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Press, New York). Alternatively, antigen-specific B cells were isolated directly from immunized VELOCIMMUNE® mice and screened using standard techniques including those described herein to obtain human antibodies specific for the antigen of interest.

Serum titer determination. To monitor the animal anti-antigen serological response, serum samples were collected approximately 10 days after each boosting, and titers were determined using an antigen-specific ELISA. Briefly, Nunc MaxiSorp™ 96-well plates were coated with 2 μg/ml antigen at 4° C. overnight and blocked with bovine serum albumin (Sigma, St. Louis, Mo.). Serum samples of serial 3-fold dilution were allowed to bind to the plate for 1 hour at room temperature. The plates were then washed with PBS containing 0.05% Tween-20, and HRP conjugated goat anti-mouse Fc (Jackson Immuno Research Laboratories, Inc., West Globe, PA) for each total IgG titer was used. Alternatively, binding IgG was detected using a biotin-labeled isotope-specific or light chain-specific polyclonal antibody (SouthernBiotech Inc.) for isotope specific titer. In the case of the biotin-labeled antibody, after washing the plate, HRP-conjugated streptabine (Pierce, Rockford, IL) was added. All plates were developed using a colorimetric substrate such as BD OptEIA® (BD Biosciences Pharmingen, San Diego, CA). After stopping the reaction with 1M phosphoric acid, the optical absorbance at 450 nm was recorded and the data were analyzed using Prism™ software from Graph Pad. To get a double background signal The required dilution is defined as the titer.

In one experiment, VELOCIMMUNE® mice were immunized with the human interleukin-6 receptor (hIL-6R). A representative set of serum titers for wild-type mice immunized with VELOCIMMUNE® and hIL-6R are shown in Figures 10A and 10B.

VELOCIMMUNE® and wild-type mice initiated a strong response to IL-6R with a similar titer range (FIG. 10A ). Several mice from the VELOCIMMUNE® and wild-type cohort reached a maximum response after a single antigen boost. These results indicate that the strength and kinetics of the immune response to this antigen are similar in VELOCIMMUNE® and wild-type mice. This antigen specific antibody response was further analyzed to investigate the specific isotope of the antigen specific antibody found in the serum. Both the VELOCIMMUNE® and wild-type groups primarily elicit an IgG1 response (FIG. 10B ), suggesting that the class switching during humoral responses is similar in each type of mouse.

Determination of the affinity of antibody binding to the antigen in solution. An ELISA based solution competition assay was typically designed to determine antibody-binding affinity for an antigen.

Briefly, the antibodies in the acclimatization medium were premixed with serial dilutions of the antigenic protein ranging from 0 to 10 mg/ml. Thereafter, a solution of the antibody and antigen mixture was incubated at room temperature for 2 to 4 hours to reach binding equilibrium. Then, the amount of free antibody in the mixture was measured using a quantitative sandwich ELISA. A 96-well Maxisorb (trade name) plate (VWR, West Chester, PA) was coated with 1 µg/ml of antigenic protein in PBS solution overnight at 4°C, followed by BSA non-specific blocking. Then, the antibody-antigen mixture solution was transferred to this plate and incubated for 1 hour. Thereafter, the plate was washed with washing buffer, and the plate-bound antibody was detected with HRP-conjugated goat anti-mouse IgG polyclonal antibody reagent (Jackson Immuno Research Lab), and BD OptEIA (trade name) (BD Biosciences Pharmingen, San Diego, CA) It was developed using the same colorimetric substrate. After stopping the reaction with 1M phosphoric acid, the optical absorbance at 450 nm was recorded and the data analyzed using Prism™ software from Graph Pad. The dependence of the signal on the concentration of antigen in solution was analyzed with a four parameter fit assay and reported as _{IC 50} , the antigen concentration required to achieve a 50% reduction in signal from the antibody sample in the absence of antigen in solution.

In one experiment, VELOCIMMUNE® mice were immunized with hIL-6R (described above). Figures 11A and 11B show a representative set of affinity measurements for anti-hIL6R antibodies from VELOCIMMUNE® and wild-type mice.

After administration of the third antigen boosting to the immunized mice, serum titers were determined by ELISA. Splenocytes were isolated from selected wild-type and VELOCIMMUNE® mouse cohorts and fused with Ag8.653 myeloma cells to form hybridomas and grown under selection (described above). Of the 671 anti-IL-6R hybridomas produced, 236 were found to express antigen-specific antibodies. Antibody affinity for the antigen was determined using a solution competition ELISA using media harvested from antigen positive wells. Antibodies derived from VELOCIMMUNE (registered trademark) mice exhibited a wide range of affinity for antigen-binding in solution (Fig. 11A). Moreover, 49 of 236 anti-IL-6R hybridomas were found to block IL-6 from binding to the receptor in an in vitro bioassay (data not shown). Additionally, these 49 anti-IL-6R blocking antibodies showed a range of high solution affinity similar to blocking antibodies derived from parallel immunization of wild-type mice (FIG. 11B ).

Example 7. Construction of mouse ADAM6 targeting vector

Targeting vectors for insertion of mouse ADAM6a and ADAM6b genes into humanized heavy chain sites were constructed using VELOCIGENE (registered trademark) genetic engineering technology (see above), and Dr. Fred Alt (Havard University) The bacterial artificial chromosome (BAC) 929d24 obtained from was modified. A genomic fragment comprising a hygromycin cassette and mouse ADAM6a and ADAM6b genes for targeted deletion of the human ADAM6 pseudogene (hADAM6ψ) located between _{the human V H} 1-2 and V _H 6-1 gene segments of the humanized heavy chain locus. 929d24 BAC DNA was engineered to contain (Figure 12).

Initially, a genomic fragment comprising the mouse ADAM6b gene, about 800 bp of the upstream (5') sequence and about 4800 bp of the downstream (3') sequence, was subcloned from the 929d24 BAC clone. A second genomic fragment comprising the mouse ADAM6a gene, about 300 bp of the upstream (5') sequence and about 3400 bp of the downstream (3') sequence, was isolated from the 929d24 BAC clone and subcloned. Two genomic fragments containing mouse ADAM6b and ADAM6a genes were ligated to flanking hygromycin cassettes with Frt recombination sites on both sides to generate a targeting vector (mouse ADAM6 targeting vector, Fig. 20; SEQ ID NO: 3). Different restriction enzyme sites were engineered to the 5'end of the targeting vector after the mouse ADAM6b gene and the 3'end after the mouse ADAM6a gene (bottom of Fig. 12) for ligation to the humanized heavy chain site.

Containing the replacement of the mouse heavy chain site by the human heavy chain site, comprising a human ADAM6 pseudogene located between _{the human V H} 1-2 and V _H 6-1 gene segments of the humanized site for subsequent ligation of the mouse ADAM6 targeting vector. A separate variant was made with the BAC clone (Figure 13).

Briefly, the _{3'position of the human V H} 1-2 gene segment (5' with respect to hADAM6ψ) and the 5'position of the human V _H 6-1 gene segment (3' with respect to hADAM6ψ; see middle of FIG. 13). A neomycin cassette with lox P recombination sites on both sides was engineered to contain the homology segment containing the human genomic sequence in. The location of the insertion site of this targeting construct is at about 1.3 kb 5'and about 350 bp 3'of the gene on human ADAM6. The targeting construct also contains the same restriction sites as the mouse ADAM6 targeting vector to allow subsequent BAC ligation between the mouse ADAM6 targeting vector and the modified BAC clone containing the deletion of the human ADAM6 pseudogene.

After digestion of BAC DNA from both constructs, genomic fragments were ligated together to construct an engineered BAC clone comprising a humanized heavy chain site comprising an ectopically located genomic sequence comprising mouse ADAM6a and ADAM6b nucleotide sequences. The final targeting construct for deletion of the human ADAM6 gene in the humanized heavy chain locus and insertion of the mouse ADAM6a and ADAM6b sequences in ES cells is from 5'to 3', the human genome sequence _{3'of the human V H 1-2 gene segment.} 5'genomic fragment comprising about 13 kb of, about 800 bp of the mouse genomic sequence downstream of the mouse ADAM6b gene, about 4800 bp of the mouse genomic sequence upstream of the mouse ADAM6b gene, the 5'Frt site, hygro of hygromycin cassette, 3'Frt region, mouse ADAM6a about 300bp of the mouse genomic sequence in the downstream of the gene, ADAM6a mouse gene, the mouse ADAM6a approximately 3400bp and human V _H gene segment 6-1 of the mouse genomic sequence in the upstream of the gene A 3'genomic fragment (bottom of Figure 13) comprising about 30 kb of human genomic sequence 5'was included.

BAC clone engineered to electroporate mouse ES cells containing humanized heavy chain sites with a generated modified ES cell containing ectopically located mouse genomic sequences comprising mouse ADAM6a and ADAM6b sequences within a humanized heavy chain locus (described above) Was used. Positive ES cells containing ectopic mouse genomic fragments within humanized heavy chain sites were identified by quantitative PCR assay using a TAQMAN® probe (Lie, YS and Petropoulos, CJ (1998) Advances in quantitative PCR technology. : 5'nuclease assays.Curr Opin Biotechnol 9(1):43-48). Using primers and probes located between the modified regions, the upstream and downstream regions outside the modified part of the humanized heavy chain site were identified by PCR to confirm the presence of the hygromycin cassette and the ectopic mouse genomic sequence within the humanized heavy chain site. . The nucleotide sequence according to the upstream insertion point included the following representing the human heavy chain genomic sequence upstream of the insertion point, and an I-Ceu I restriction site (contained within the paragraphs below) successively linked to the mouse genomic sequence present at the insertion point: (CCAGCTTCAT TAGTAATCGT TCATCTGTGG TAAAAAGGCA GGATTTGAAG CGATGGAAGA TGGGAGTACG GGGCGTTGGA AGACAAAGTG CCACACAGCG CAGCCTTCGT CTAGACCCCC GGGCTAACTA TAACGGTCCT AAGGTAGCGA GGGCTAACTA TAACGGTCCT AAGGTAGCGA GGGGCTAACTA TAACGGTCCT AAGGTAGCGA G) GGGATCCCCCCCCCCACGCGAGGTCTGACTGCTGCACGAGCGATGCTAGGTTGCTG The nucleotide sequence according to the downstream insertion point at the 3'end of the targeted region is the following representing the mouse genomic sequence and the PI-Sce I restriction (contained within the paragraphs below) connected in succession with the human heavy chain genomic sequence downstream of the insertion point. Sites included: (AGGGGTCGAG GGGGAATTTT ACAAAGAACA AAGAAGCGGG CATCTGCTGA CATGAGGGCC GAAGTCAGGC TCCAGGCAGC GGGAGCTCCA CCGCGGTGGC GCCATTTCAT TACCTCTTTC TCCGCACCCG ACATAGATAAAGCTTTC TCCGCACCCG ACATAGATAAAGCTT) ATCCCCCACC AAGCGCAAGCCGCATGCATGCAAGCAAGCCGAGCATGCAAGCAAGCCGAGCATGCAAGCAAGCCGAGCACGT

The targeted ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos by the VELOCIMOUSE® mouse manipulation method (see, e.g., US 7,6598,442, 7,576,259, 7,294,754). I did. Humanized heavy chain comprising an ectopic mouse genomic sequence comprising mouse ADAM6a and ADAM6b sequences by genotyping using modification of an allele assay (Valenzuela et al ., 2003) to detect the presence of mouse ADAM6a and ADAM6b genes within the humanized heavy chain locus Mice holding spots were identified.

Mice bearing humanized heavy chain loci containing mouse ADAM6a and ADAM6b genes were grown as FLPe deletion mouse strains ( e.g., Rodriguez, CI et al. (2000) High-efficiency deleter mice show that FLPe is an alternative to Cre- lox P. Nature Genetics 25:139-140), such as any Frtified hygromycin cassette introduced by a targeting vector that was not removed from the ES cell or from the embryo was removed. Optionally, the hygromycin cassette was retained in mice.

Pup was genotyped, and pup heterozygous for a humanized heavy chain locus comprising an ectopic mouse genomic fragment comprising mouse ADAM6a and ADAM6b sequences was selected to characterize mouse ADAM6 gene expression and fertility.

Example 8. Identification of ADAM6 rescue mice

Flow cytometer . 3 mice and humans at 25 weeks old homozygous for human heavy and human κ light chain variable loci (H/κ) for identification and analysis of lymphocyte cell populations by FAC in the BD LSR II system (BD Bioscience) Three mice at 18-20 weeks of age homozygous for a human heavy chain and a human κ light chain with ectopic mouse genomic fragments encoding the mouse ADAM6a and ADAM6b genes within both alleles of the heavy chain locus (H/κ-A6). Sacrificed. Lymphocytes were gated for specific cell lineages and analyzed for progression across various B cell generators. Tissues collected from animals included blood, spleen and bone marrow. Blood was collected in BD microvessel tubes with EDTA (BD Biosciences). Bone marrow was collected from the femur by washing with complete RPMI medium supplemented with fetal bovine serum, sodium dermatate, HEPES, 2-mercaptoethanol, non-essential amino acids and gentamicin. Red blood cells from the blood, spleen and bone marrow populations were lysed with an ammonium chloride-based lysis buffer (eg , ACK lysis buffer) and then washed with complete RPMI medium.

For staining of cell populations, 1× ^{10 6} cells from various tissue sources were incubated with anti-mouse CD16/CD32 (2.4G2, BD Biosciences) for 10 minutes on ice, followed by one or a combination of the following antibody cocktails. Labeled for 30 minutes on ice.

Bone marrow: anti-mouse FITC-CD43 (1B11, BioLegend), PE-c kit (2B8, BioLegend), PeCy7-IgM (II/41, eBioscience), PerCP-Cy5.5-IgD (11-26c.2a, BioLegend) , APC-eFluor780-B220 (RA3-6B2, eBioscience), A700-CD19 (1D3, BD Biosciences).

Peripheral blood and spleen: anti-mouse FITC-κ (187.1, BD Biosciences), PE-λ (RML-42, BioLegend), PeCy7-IgM (II/41, eBioscience), PerCP-Cy5.5-IgD (11-26c .2a, BioLegend), APC-CD3 (145-2C11, BD), A700-CD19 (1D3, BD), APC-eFluor780-B220 (RA3-6B2, eBioscience). After incubation with the labeled antibody, the cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on an LSRII flow cytometer and analyzed with FlowJo. Results from representative H/κ and H/κ-A6 mice are shown in Figures 14-18.

These results show that B cells from H/κ-A6 mice progress through the B cell generator in a similar manner to H/κ mice in the bone marrow and peripheral compartments, and when they enter the periphery, they exhibit a normal pattern of maturation. H/κ-A6 mice show an increased CD43 ^int CD19 ⁺ cell population compared to H/κ mice (FIG. 16B ). This can show accelerated IgM expression from humanized heavy chain loci comprising ectopic mouse genomic fragments comprising mouse ADAM6a and ADAM6b sequences in H/κ-A6 mice. Peripheral, the B and T cell populations of H/K-A6 mice are normal and appear to be similar to H/K mice.

Identification of testicular morphology and sperm. To determine whether the infertility of mice with humanized immunoglobulin heavy chain variable sites is due to testis and/or spermatogenic binding, the testis morphology and sperm content of the epididymis were examined.

Briefly, group (Group 1: mouse homozygous for human heavy chain and κ light chain variable locus, mADAM6 ^-/- ; Group 2: mouse heterozygous for human heavy chain variable locus and κ light chain variable locus , mADAM6 ^+/- ), testes from 2 groups of 5 mice were excised and weighed, with epididymis intact. Thereafter, the specimen was fixed, embedded in paraffin, excised, and stained with hematoxylin and eosin (HE) staining. Testicular sections (2 testes per mouse, total 20) were examined for sperm generation traces and morphological defects, and epididymal sections were examined for the presence of sperm.

In this experiment, no difference in testicular weight or morphology was observed between ^{mADAM6 -/-} mice and mADAM6 ^{+/- mice.} Sperm were observed in all genotypes of both testis and epididymis. These results demonstrate that the absence of the mouse ADAM6a and ADAM6b genes does not result in a detectable change in testicular morphology, and sperm are produced in mice in the presence and absence of these two genes. The male ADAM6 ^-/- mouse's fertility defect is not due to low sperm production.

Sperm motility and movement. Mice lacking other ADAM gene family members are infertile due to defects in sperm motility or migration. Sperm motility is defined as the ability of sperm to pass from the uterus to the fallopian tube and is usually required for fertilization in mice. To determine if deletion of mouse ADAM6a and ADAM6b affects this process, sperm migration in ^{mADAM6 -/- mice was evaluated.} Sperm motility was also investigated.

Briefly, (1) mice heterozygous for human heavy chain variable locus and homozygous for human κ light chain variable locus (ADAM6 ^+/- ); (2) mice homozygous for human heavy chain variable locus and homozygous for human κ light chain variable locus (ADAM6 ^-/- ); (3) mice homozygous for human heavy chain variable locus and homozygous for wild-type κ light chain (ADAM6 ^-/- mk); And (4) sperm were obtained from the testes of wild-type C57 BL/6 mice (WT). No significant abnormalities in sperm count or total sperm motility were observed. For all mice, cumulus proliferation was observed, indicating that each sperm sample could penetrate the cumulus cells and bind to the in vitro zona pellucida. This result demonstrates that ADAM6 ^-/- mice have sperm that can infiltrate the egg bulb and bind to the zona pellucida.

Described above were performed in the laboratory mouse oocytes with sperm from the mouse modified (IVF) in bar. On the day after IVF, ^{the number of embryos divided into ADAM6 -/-} was slightly smaller, and there were fewer sperm bound to the egg. This result demonstrates that once exposed to the egg, ^{sperm from ADAM6 -/-} mice can penetrate the egg bulb and bind to the zona pellucida.

In another experiment, the ability of sperm from ^{ADAM6 -/-} mice to migrate from the uterus through the fallopian tube in a sperm migration assay was determined.

Briefly, a first group of 5 hyperovulated female mice was set up with ^{5 ADAM6 -/- males.} A second group of 5 overovulated mice was set up with ^{5 ADAM6 +/- males.} Intercourse mating pairs were observed, and after 5 to 6 hours of intercourse, the uterus and attached fallopian tubes were removed from all females and washed for analysis. The flushing solution was checked against eggs to confirm ovulation and sperm counts were obtained. Sperm migration was evaluated in two different ways. Initially, both fallopian tubes were removed from the uterus, washed with saline and any sperm identified were counted. The presence of eggs as evidence of ovulation is also mentioned. Second, the fallopian tubes were left attached to the uterus, and both tissues were fixed, embedded in paraffin, incised, and stained (described above). Sections were examined for the presence of sperm in both the uterus and fallopian tubes.

In ^{the case of 5 females mated with 5 ADAM6 -/-} males, very few sperm were identified in the flushing solution from the fallopian tube. The flushing solution from the fallopian tubes of 5 females mated with 5 ADAM6 ^+/- males is about 25 than that present in the flushing solution from the fallopian tubes of 5 females mated with 5 ^{ADAM6 -/- males.} Sperm levels were 10 times to 30 times higher (average, n = 10 fallopian tubes).

Histological sections of the uterus and fallopian tubes were prepared. Sections were examined for the presence of sperm in the uterus and fallopian tubes (gull ridge). Examination of the fallopian tubes and histological sections of the ^{uterus indicated that in the case of female mice mated with ADAM6 -/-} mice, sperm were observed in the uterus, not in the fallopian tubes. Additionally, ^{sections from female mated with ADAM6 -/-} mice indicate that no sperm are observed at the uterine fallopian tube junction (UTJ). In ^{sections from females mated with ADAM6 +/-} mice, sperm were identified in the UTJ and fallopian tubes.

These results indicate that mice lacking the ADAM6a and ADAM6b genes produce sperm exhibiting migration defects in vivo. In all cases, sperm are observed in the uterus, indicating that ovulation and sperm release are clearly normal, but there is little or no sperm in the fallopian tube after ovulation as measured by sperm count or histological observation. These results indicate that mice lacking the ADAM6a and ADAM6b genes produce sperm, indicating a lack of ability to migrate from the uterus to the fallopian tube. This defect clearly leads to infertility as sperm cannot cross the uterine-tubal junction into the fallopian tube into which the egg is fertilized. Taken together, all these results, combined, support the hypothesis that sperm with normal motility directs them to move out of the uterus via the fallopian tube junction and fallopian tubes, thus assisting in accessing the egg to achieve a fertilization event. The mechanism by which ADAM6 achieves this can be either directly by the action of the ADAM6 protein or by systemic expression with other proteins, such as other ADAM proteins, in sperm cells as described below.

ADAM gene family expression. It is known that the ADAM protein complex exists as a complex for the surface of mature sperm. Mice lacking other ADAM gene family members lose this complex as sperm maturity, and show a decrease in multiple ADAM proteins in mature sperm. Expression of other ADAM gene family members by analyzing western blots of protein extracts from testis (immature sperm) and epididymis (mature sperm) to determine whether defects in the ADAM6a and ADAM6b genes affect other ADAM proteins in a similar manner. The level was determined.

In this experiment, ^{protein extracts from 4 ADAM6 -/-} and 4 ADAM6 ^+/- mice were analyzed. The results indicate that the expression of ADAM2 and ADAM3 is not affected in the testis extract. However, both ADAM2 and ADAM3 decreased dramatically in epididymis extract. This indicates that the absence of ADAM6a and ADAM6b in the sperm of ^ADAM6-/- mice may have a direct effect on the expression and possibly function of other ADAM proteins as sperm maturation (eg, ADAM2 and ADAM3). This suggests that ADAM6a and ADAM6b are part of the ADAM protein complex on the surface of sperm, which may be important for proper sperm migration.

Example 9. Utilization of human heavy chain variable gene in ADAM6 rescue mice

^{Mouse ADAM6a and ADAM6b genes lack (mADAM6 -/-} ) or contain ectopic genomic fragments encoding mouse ADAM6a and ADAM6b genes by quantitative PCR assay using a Taqman® probe (described above) ^{(ADAM6 +/+} The use of selected human heavy chain variable genes for mice homozygous for the human heavy chain and κ light chain variable locus (see Example 1) was determined.

Briefly, CD19 ^{+ B cells were purified from the spleen of mADAM6 -/-} and ADAM6 ^+/+ mice using mouse CD19 microbeads (Miltenyi Biotec), and total RNA was purified using the RNeasy™ mini kit (Qiagen). And purified. Genomic RNA was removed using treatment on an RNase-free DNase column (Qiagen). About 200 ng of mRNA was reverse transcribed into cDNA using First Stand cDNA Synthesis kit (Invitrogen), and then Tachman (trade name) universal PCR master mix (trademark) using ABI 7900 sequence detection system (Applied Biosystems). Universal PCR Master Mix) (Applied Biosystems). The relative expression of each gene was normalized to the mouse κ constant region (mCκ). Table 9 lists the sense/antisense/Tachman® MGB probe combinations used in this experiment.

In this experiment, expression of all four human V _H genes was observed in the samples analyzed. Additionally, expression levels were comparable to ^{mADAM6 -/-} and ADAM6 ^{+/+ mice.} This result shows that the human V _H genes distal to the modified sites (V _H 3-23 and V _H 1-69) and proximal to the modified sites (V _H 1-2 and V _H 6-1) are functionally expressed in combination Human heavy chains can be formed. These results show that the ectopic genomic fragment containing the mouse ADAM6a and ADAM6b sequences inserted into the human heavy chain genome sequence did not affect the V(D)J recombination of the human heavy chain gene segment in situ, and the mouse did not affect the human heavy chain gene segment. It indicates that it can be recombined in a normal manner to produce a functional heavy chain immunoglobulin protein.

Example 10. Deletion of mouse immunoglobulin light chain site

Velocigen® technology (e.g., US Pat. No. 6,586,251 and Valenzuela et al. (2003) to modify mouse genomic bacterial artificial chromosome (BAC) libraries to inactivate mouse κ and λ light chain sites. ) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659]) to create various targeting constructs.

Deletion of the mouse λ light chain site. DNA from mouse BAC clone RP23-135k15 (Invitrogen) was modified by homologous recombination to inactivate the endogenous mouse λ light chain site through targeted deletion of the Vλ-Jl-Cλ gene cluster (FIG. 20 ).

Briefly, a neomycin cassette having a lox P site on both sides with a 5'mouse homology section comprising the 5'sequence of the Vλ1 gene segment and a 3'mouse homology section comprising the 3'sequence of the Cλ1 gene segment. The containing targeting vector was used to delete the entire proximal cluster containing the Vλ1-Jλ3-Cλ3-Jλ1-Cλ1 gene segment in a single targeting event (FIG. 20, targeting vector 1).

Vλ2-Jλ2-, except that the targeting construct contains a 5'mouse homology section comprising the 5'sequence of the Vλ2 gene segment and a 3'mouse homology section comprising the 5'sequence for the endogenous Cλ2 gene segment. A second targeting construct was prepared to accurately delete the distal endogenous mouse λ gene cluster containing Cλ2-Jλ4-Cλ4 (Fig. 20, targeting vector 2). Thus, the second targeting construct correctly deleted Vλ2-Jλ2, leaving Cλ2-Jλ4-Cλ4 intact at the endogenous mouse λ site. ES cells containing an inactivated endogenous λ site (described above) were identified by karyotyping and screening methods known in the art (eg, Tachman®). Thereafter, DNA was isolated from the modified ES cells and treated with CRE recombinase to mediate the deletion of the proximal targeting cassette containing the neomycin marker gene, leaving only a single lox P site at the deletion point (Fig. 20, bottom). .

Deletion of the mouse κ light chain site. Several targeting constructs were made using a similar method described above to modify DNA from mouse BAC clones RP23-302g12 and RP23-254m04 (Invitrogen) by homologous recombination to inactivate the mouse κ light chain site in the two-step process. (Fig. 21).

Briefly, using a targeting vector comprising a hygromycin-thymidine kinase (hyg-TK) cassette containing a single lox P site 3'to the hyg-TK cassette, the endogenous mouse κ light chain site was The Jκ gene segment (1-5) was deleted (Fig. 21, Jκ targeting vector). The homology section used to make this targeting vector included the mouse genomic sequences that are 5'and 3'of the endogenous mouse JK gene segment. In the second targeting event, a second targeting vector was prepared to delete a portion of the mouse genomic sequence upstream (5') to the most distal endogenous mouse Vκ gene segment (Figure 21, Vκ targeting vector). This targeting vector contained a neomycin cassette and an inverted lox 511 site, a lox P site. The homology section used to make this targeting vector contained the mouse genomic sequence upstream of the most distal mouse VK gene segment. Targeting vectors were used in a sequential manner (ie, Jκ followed by Vκ) to target DNA in ES cells. ESs carrying double targeting chromosomes (i.e., a single endogenous mouse κ site targeted with both targeting vectors) were identified by karyotyping and screening methods known in the art (eg , Tachman®). Thereafter, DNA was isolated from the modified ES cells and treated with Cre recombinase to mediate deletion of both the selection cassette and the endogenous mouse Vκ gene segment , leaving the two juxtaposed lox sites in opposite orientations to each other (FIG. 21 , Bottom; SEQ ID NO: 59).

Thus, the targeting vectors described below were used to progressively insert unrearranged human λ germline gene segments in the correct manner to create two modified endogenous light chain sites (κ and λ) comprising intact enhancers and constant regions.

Example 11. Replacement of mouse light chain locus by human lambda light chain small locus

Multiple targeting vectors were engineered to progressively insert human λ gene segments into endogenous mouse κ and λ light chain sites using a similar method described above. A number of independent initial modifications were made to the endogenous light chain locus to prepare a chimeric light chain locus comprising hVλ and Jλ gene segments operatively linked to a mouse light chain constant gene and enhancer, respectively.

Human λ small locus containing 12 human Vλ and 1 human Jλ gene segments. A human BAC clone called RP11-729g4 (Invitrogen) was used to engineer a series of initial targeting vectors to contain a first 12 consecutive human Vλ gene segments and hJλ1 gene segments or four hJλ gene segments from cluster A origin. . 22A and 22B show targeting vectors constructed to prepare initial inserts of human λ light chain gene segments at mouse λ and κ light chain sites, respectively.

For the first set of initial targeting vectors, a 124,125 bp DNA fragment from a 729g4 BAC clone comprising a 12 hVλ gene segment and an hJλ1 gene segment was 3′ downstream of the hJλ1 gene segment (3′) for ligation of the mouse homology segment. ) Was engineered to include a PISceI site 996bp. Two different sets of homology segments were used for ligation into this human fragment; One set of homology divisions contained endogenous mouse λ sequences from the 135k15 BAC clone (FIG. 22A ), and another set 5 of the mouse VK and JK gene segments from mouse BAC clones RP23-302g12 and RP23-254m04, respectively. Endogenous κ sequences were included in'and 3'(FIG. 22B).

For the 12/1-λ targeting vector (FIG. 22A ), the PI-SceI site was the 5′ end of a 27,847 bp DNA fragment containing the mouse Cλ2-Jλ4-Cλ4 and the enhancer 2.4 of the modified mouse λ site described in Example 10. It was operated in. The about 28 kb mouse fragment is from 5'to 3', the hJλ1 gene segment, the 996 bp human λ sequence at 3'of the hJλ1 gene segment, the 1229 bp mouse λ sequence at 5'to the mouse Cλ2 gene, the mouse Cλ2 gene, and It was used as a 3'homology section by ligation to an about 124 kb human λ fragment, creating a 3'junction comprising the remainder of the about 28 kb mouse fragment. The upstream (5') from the human Vλ3-12 gene segment contains 23,792 bp of mouse genomic DNA corresponding to the 5'sequence of the endogenous mouse λ locus, with an additional 1456 bp of human before the beginning of the 5'mouse homology section. λ sequence. There is a neomycin cassette with an Frt site on both sides between the 5'homology segment and the beginning of the human λ sequence.

Thus, the 12/1-λ targeting vector is a 5'homology segment comprising a mouse λ genomic sequence of about 24 kb at 5'of the endogenous λ site, 5'Frt site, neomycin cassette, from 5'to 3', A mouse of about 28 kb comprising a 3'Frt site, a human genomic λ sequence of about 123 kb comprising a first 12 contiguous hVλ gene segments and an hJλ gene segment, a PI-SceI site, and an endogenous Cλ2-Jλ4-Cλ4 gene segment It contains a genomic sequence, a mouse enhancer 2.4 sequence, and a 3'homology segment comprising an additional mouse genomic sequence downstream (3') of enhancer 2.4 (FIG. 22A ).

In a similar manner, the 12/1-κ targeting vector (FIG. 22B ), except that a mouse homology section containing the mouse κ sequence is used so that targeting to the endogenous κ site can be achieved by homologous recombination. The same about 124 human λ fragments were used. Thus, the 2/1-κ targeting vector is a 5'homology segment comprising a mouse genomic sequence of about 23 kb at 5'of an endogenous κ site, from 5'to 3', an I-CeuI site, a 5'Frt site, About 124 kb of human genomic λ sequence comprising a neomycin cassette, a 3'Frt site, a first 12 consecutive hVλ gene segments and an hJλ1 gene segment, a PI-SceI site and an endogenous Cκ gene, Eκi and Eκ3'. It contains a 28 kb mouse genomic sequence and a 3'homology segment comprising an additional mouse genomic sequence downstream (3') of Eκ3' (Fig. 22B, 12/1-κ targeting vector).

Endogenous mouse light chain constant genes and enhancers (Cκ or Cλ2 and Eκi/Eκ3' or Enh2.4/Enh3.1) that induce the formation of chimeric λ light chains upon recombination by homologous recombination by one of these two initial targeting vectors. A modified mouse light chain locus (κ or λ) comprising an operatively linked 12hVλ gene segment and an hJλ1 gene segment was generated.

Human λ small locus with 12 human Vλ and 4 human Jλ gene segments. In another approach to add diversity to the chimeric λ light chain site, the third initial targeting vector is the first 12 consecutive human Vλ gene segments from cluster A and the hJλ1, 2, 3 and 7 gene segments to the mouse κ light chain site. Manipulated to insert (Fig. 22B, 12/4-κ targeting vector). DNA segments containing the hJλ1, Jλ2, Jλ3 and Jλ7 gene segments were prepared by neonatal DNA synthesis (Integrated DNA Technologies), which were respectively Jλ gene segments and two regions directly at 5′ and 3′ of each Jλ gene segment. It contains about 100 bp of human genomic sequence of multiple origins. The PI-SceI site was engineered into the 3'end of a DNA fragment of about 1 kb and ligated to a chlororamphenicol cassette. The homology section was PCR amplified from the human λ sequence at the 5'and 3'positions for the hJλ1 gene segment of human BAC clone 729g4. Homologous recombination with an intermediate targeting vector was performed upstream of a human Vλ3-12 gene segment with a neomycin cassette flanking the Frt region that also contains an I-CeuI site 5'to the 5'Frt site (5' ) In a modified 729g4 BAC clone already targeted. The double-targeted 729g4 BAC clone is a fragment of about 123 kb comprising the first 12 hVλ gene segment, from 5'to 3', I-CeuI site, 5'Frt site, neomycin cassette, 3'Frt site, human A fragment of about 1 kb comprising the Jλ1, 2, 3 and 7 gene segments, a PI-SceI site, and a chlororamphenicol cassette. The intermediate targeting vector was digested with ICeuI and PI-SceI and subsequently ligated to the modified mouse BAC clone (described above) to generate a third targeting vector.

This ligation creates a third targeting vector for inserting the human λ sequence into the endogenous κ light chain site, and the vector is 5'to 3', containing a genomic sequence of about 23 kb in the 5'of the endogenous mouse κ site. 'Mouse homology section, I-CeuI site, 5'Frt site, neomycin cassette, 3'Frt site, a fragment of about 123 kb containing the first 12 hVλ gene segment and hJλ1, 2, 3 and 7 gene segments. 3 comprising a fragment of about 1 kb comprising, a PI-SceI site and an endogenous mouse Cκ gene, a mouse genomic sequence of about 28 kb comprising Eκi and Eκ3′ and a mouse genomic sequence additional to downstream (3′) of Eκ3′. 'Includes homology section (Figure 22, 12/1-κ targeting vector). Homologous recombination with a third targeting vector is a modified comprising a 12 hVλ gene segment and four hJλ gene segments, operably linked to an endogenous mouse Cκ gene, which upon recombination forms a chimeric human λ/mouse κ light chain. A mouse κ light chain site was created.

Human λ small site with integrated human κ light chain sequence. In a similar manner, two additional targeting vectors similar to those engineered to create initial inserts (Fig. 22B, 12/1-λ and 12/4-κ targeting vectors) into the endogenous κ light chain site of a human λ gene segment. Was engineered to progressively insert human λ light chain gene segments using a uniquely designed targeting vector comprising contiguous human λ and κ genomic sequences. This targeting vector was constructed to contain approximately 23 kb of human κ genomic sequence naturally located between the human Vκ4-1 and Jκ1 gene segments. This human κ genomic sequence was specifically located between the human Vλ and human Jλ gene segments in these two additional insertion vectors (Fig. 22B, 12(κ)-κ and 12(κ)4-κ targeting vectors).

Both targeting vectors containing the human κ genomic sequence were prepared using the modified RP11-729g4 BAC clone described above (FIG. 24 ). This modified BAC clone was targeted with a spectinomycin selection cassette with NotI and AsiSI restriction sites on both sides (FIG. 24, upper left). Homologous recombination by spectinomycin cassette is from 5'to 3', I-CeuI site, 5'Frt site, neomycin cassette, 3'Frt site, about 123 kb including the first 12 hVλ gene segment. Fragment, a double targeted 729g4 BAC clone containing a NotI site, a spectinomycin cassette and an AsiSI site at about 200 bp downstream (3′) to the nonamer sequence of the hVλ3-1 gene segment was generated. A separate human BAC clone (CTD-2366j12) containing a human κ sequence was contained within a modified 729g4 BAC clone that was double-targeted by manipulating a restriction site at a location between the hVκ4-1 and hJκ1 gene segments at two separate time points. The hVλ gene segment was targeted to enable subsequent cloning of a fragment of about 23 kb for ligation (Fig. 24, upper right).

Briefly, the 2366j12 BAC clone is about 132 kb in size and has a human κ genome sequence downstream of the hVκ gene segments 1-6, 1-5, 2-4, 7-3, 5-2, 4-1, and Vκ gene segments. , hJK gene segments 1-5, hCK and about 20 kb of additional genomic sequence of the human κ site. This clone was first targeted with a targeting vector comprising a hygromycin cassette with an Frt site on both sides and a NotI site downstream (3′) of the 3′ Frt site. The homology division for this targeting vector includes human genomic sequences at 5'and 3'of the Vκ gene segment in the BAC clone, so when homologous recombination by this targeting vector the Vκ gene segment is deleted and the NotI site is hVκ 4-1. It is engineered about 133 bp downstream of the gene segment (Figure 24, top right). This modified 2366j12 BAC clone was independently targeted with two targeting vectors at the 3'end to identify the hJλ1 gene segment, the PI-SceI site and the AsiSI site, or the four hJλ gene segments (see above), the PI-SceI site, and the AsiSI site. The hJκ gene segment with a chlororamphenicol cassette that also contains the containing human λ genomic fragment is deleted (FIG. 24, upper right). The homology divisions for these two similar targeting vectors include sequences at 5'and 3'of the hJK gene segment. Homologous recombination by these second targeting vectors and modified 2366J12 BAC clones, from 5'to 3', 5'Frt site, hygromycin cassette, 3'Frt site, NotI site, Vκ4-1 and Jκ1 gene segments 22,800bp genomic fragment of the human κ site including the intergenic region, hJλ1 gene segment or human λ genome fragment including hJλ1, Jλ2, Jλ3 and Jλ7, PI-SceI site, and a chlororamphenicol cassette. A double targeted 2366j12 clone was generated (Figure 24, top right). The two final targeting vectors to make two additional modifications were achieved by two ligation steps using double targeted 729g4 and 2366j12 clones.

The double-targeted 729g4 and 2366j12 clones were digested with NotI and AsiSI to approximately 23 kb of the human κ site, including one fragment containing the neomycin cassette and hVλ gene segments, respectively, and the intergenic region between the VK4-1 and JK1 gene segments. Genomic fragments of, hJλ1 gene segments or genomic fragments including the hJλ1, Jλ2, Jλ3 and Jλ7 gene segments, PI-SceI sites, and other fragments including the chlororamphenicol cassette were generated. The ligation of these fragments is from 5'to 3', a genomic fragment comprising an hVλ gene segment, a human κ genome sequence between the Vκ4-1 and Jκ1 gene segments, an hJλ1 gene segment, or hJλ1, Jλ2, Jλ3, and Jλ7 gene segments, Two unique BAC clones were generated containing the PI-SceI site and the chlororamphenicol cassette (Figure 24, bottom). Thereafter, these new BAC clones were digested with I-CeuI and PI-SceI to release a unique fragment containing the upstream neomycin cassette and contiguous human λ and κ sequences, from 5'to 3', 5'of the endogenous κ site. Mouse genomic sequence, I-CeuI site, 5'Frt site, neomycin cassette, 3'Frt site, hVλ gene segment (3-12 to 3-1), NotI site at about 200 bp downstream of Vλ3-1 , About 23 kb of human κ sequence naturally found between human Vκ4-1 and Jκ1 gene segments, hJλ1 gene segments or genomic fragments comprising hJλ1, Jλ2, Jλ3 and hJλ7 gene segments, mouse Eκi, mouse Cκ gene and Eκ3 The modified mouse BAC clone 302g12 containing 'was ligated (Fig. 22, 12hVλ-VκJκ-hJλ1 and 12hVλ-VκJκ-4hJλ targeting vectors). Homologous recombination with both of these targeting vectors, upon recombination, is one or four hJλ operably linked to the 12hVλ gene segment, the human κ genomic sequence and the endogenous mouse Cκ gene, which, upon recombination, forms a chimeric human l/mouse κ light chain Two distinct modified mouse κ light chain sites containing gene segments were generated.

Example 12. Manipulation of additional human Vλ gene segments into human λ light chain small loci

Additional hVλ gene segments were added independently to each of the initial modifications described in Example 11 using similar targeting vectors and methods (Fig. 23A, +16-λ targeting vector and Fig. 23B, +16-κ targeting vector).

Introduction of 16 additional human Vλ gene segments. The upstream (5') homology segment used to construct a targeting vector to add 16 additional hVλ gene segments to the modified light chain site described in Example 112 is a mouse at 5'of the endogenous κ or λ light chain site. It contains genomic sequence. The 3'homology segment is the same for all targeting vectors and includes human genomic sequences that overlap the 5'end of the human λ sequence of the modification as described in Example 11.

Briefly, two targeting vectors were engineered for the introduction of 16 additional hVλ gene segments into the modified mouse light chain locus described in Example 11 (Figures 23A and 5B, +16-λ or +16-κ targeting vectors. ). A DNA fragment of about 172 kb from human BAC clone RP11-761113 (Invitrogen) comprising 21 contiguous hVλ gene segments of cluster A origin was transferred to 5 comprising a mouse genomic sequence 5'to the endogenous κ or λ light chain site. It was engineered with the'homology section and 3'homology section containing the human genome λ sequence. The 5'mouse κ or λ homology section used in these targeting constructs is the same 5'homology section as described in Example 11 (FIGS. 23A and 23B ). The 3'homology section includes a 53,057 bp overlap of the human genomic λ sequence corresponding to the equivalent 5'end of the human genomic λ sequence of the about 123 kb fragment described in Example 11. These two targeting vectors are 5'to 3', comprising a genomic sequence of about 23 kb at 5'of the endogenous mouse κ light chain site or about 24 kb of mouse genomic sequence 5'of the endogenous λ light chain site. Mouse homology section, 5'Frt site, hygromycin cassette, 3'Fit site, and about 53 kb overlap with the 5'end of the human λ sequence described in Example 12 and as a 3'homology section to the targeting construct. It contains a 171,457 bp human genomic λ sequence comprising 21 contiguous hVλ gene segments in action (Figs. 23A and 23B, +16-λ or +16-κ targeting vectors). Homologous recombination by these targeting vectors is an independently modified mouse κ comprising a 28hVλ gene segment and an hJλ1 gene segment operably linked to an endogenous mouse constant gene (Cκ or Cλ2), respectively, that, upon recombination, form a chimeric light chain. And λ light chain sites were generated.

In a similar manner, the +16-κ targeting vector was also used to add 16 additional hVλ gene segments to other initial modifications described in Example 11 incorporating multiple hJλ gene segments in the presence and absence of an integrated human κ sequence. Introduced (Fig. 22b). Homologous recombination by targeting vectors at the endogenous mouse κ site with other early modifications, upon recombination, results in the presence of a human Vκ-Jκ genomic sequence operably linked to the endogenous mouse Cκ gene, forming a chimeric λ-κ light chain. Or in the absence of a mouse κ light chain locus comprising 28 hVλ gene segments and hJλ1, 2, 3, and 7 gene segments.

Introduction of 12 additional human Vλ gene segments. Additional hVλ gene segments were added independently to each of the modifications described above using similar targeting vectors and methods. The final locus structure obtained from homologous recombination with a targeting vector containing an additional hVλ gene segment is shown in FIGS. 25A and 25B.

Briefly, a targeting vector was engineered for the introduction of 12 additional hVλ gene segments into the modified mouse κ and λ light chain sites described above (Figs. 23A and 23B, +12-λ or 12-κ targeting vectors). A DNA fragment of 93,674 bp from human BAC clone RP11-22I18 (Invitrogen) comprising 12 contiguous hVλ gene segments of cluster B origin comprises a mouse genomic sequence 5'to the endogenous mouse κ or λ light chain site. It was engineered with a 5'homology section and a 3'homology section containing the human genome λ sequence. The 5'homology section used for this targeting construct is the same 5'homology section used for the addition of the 16 hVλ gene segments described above (FIGS. 23A and 23B ). The 3'homology section was prepared by engineering the PI-SceI site at about 3431 bp 5'to the human Vλ3-29P gene segment contained in the 27,468 bp genomic fragment of the human λ sequence from BAC clone RP11-761I13. This PI-SceI site contains about 94 kb of an additional human λ sequence fragment with the 5'end of the human λ sequence in a previous modification using a +16-λ or +16-κ targeting vector (Figures 23A and 23B). It serves as a ligation point for linking with overlapping approximately 27 kb human λ sequence fragments. These two targeting vectors are 5'to 3', comprising a mouse genomic sequence of about 23 kb at 5'of the endogenous κ light chain site, or about 24 kb of mouse genomic sequence 5'of the endogenous λ light chain site. 'Homology division, 5'Frt site, neomycin cassette, 3'Frt site and about 27 kb overlap with the 5'end of the human λ sequence from the insertion origin of 16 additional hVλ gene segments and 3'for this targeting construct. It contains a 121,188 bp human genomic λ sequence comprising a 16 hVλ gene segment and a PI-SceI site, serving as a homology division (Figures 23A and 23B, +12-λ or 12-κ targeting vectors). Homologous recombination by this targeting vector is a modified mouse κ and λ light chain comprising a 40 hVλ gene segment operably linked to an endogenous mouse constant gene (Cκ or Cλ2) and human Jλ1, which, upon recombination, forms a chimeric light chain. Sites were created independently (bottom of FIGS. 23A and 23B).

In a similar manner, a +12-κ targeting vector was also used to introduce 12 additional hVλ gene segments into other initial modifications that incorporate multiple hJλ gene segments in the presence or absence of an integrated human κ sequence (FIG. 22B ). . Homologous recombination by this targeting vector at the endogenous mouse κ site, including other modifications, results in the presence and absence of a human Vκ-Jκ genomic sequence operably linked to the endogenous mouse Cκ gene, which upon recombination forms a λ-κ light chain. Below, a mouse κ light chain locus was generated comprising 40 hVλ gene segments and hJλ1, 2, 3, and 7 gene segments.

Example 13. Identification of targeted ES cells bearing human λ light chain gene segments

The modified ES cells were generated to generate chimeric mice expressing the human λ light chain gene segment by electroporation of mouse ES cells using the targeted BAC DNA prepared according to the previous example. ES cells containing an insert of an unrearranged human λ light chain gene segment were confirmed by quantitative Tachman® analysis. Specific primer sets and probes include insertion of human λ sequences and related selection cassettes (obtain alleles, GOA), endogenous mouse sequences and loss of any selection cassettes (loss of alleles, LOA) and retention of flanking mouse sequences. Designed for (allele retention, AR). For each additional insertion of the human λ sequence, the presence of additional human λ sequences as well as previous primer sets and probes used to confirm retention of previously targeted human sequences using additional primer sets and probes. Was confirmed. Velocimuno describes primers and related probes used in quantitative PCR assays. Table 11 lists the combinations used to confirm the insertion for each segment of the human λ light chain gene segment in the ES cell clone.

ES cells carrying a human λ light chain gene segment are optionally transfected with a construct expressing FLP to obtain a Frt-ized neomycin cassette introduced by insertion of a targeting construct comprising a human Vλ5-52-Vλ1-40 gene segment. Removed (Figs. 23A and 23B). The neomycin cassette can optionally be removed by crossing to mice expressing the FLP recombinase (eg, US 6,774,279). Optionally, the neomycin cassette is retained in the mouse.

Example 14. Generation of mice expressing human λ light chain from endogenous light chain locus

The targeted ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos by the veloshimaus method (eg, US Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully fully described. derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1):91-99). VELOCIMICE (F0 mice fully derived from donor ES cells) independently bearing human λ gene segments is a modification of a modified allele assay (Valenzuela et al ., supra) that detects the presence of a unique human λ gene segment (Valenzuela et al., supra). Reference) was confirmed by genotyping (see above).

Use of the κ:λ light chain in mice carrying the human λ light chain gene segment. A single hJλ gene segment comprising a mouse homozygous (FIG. 23B) and a human Vκ-Jκ genomic sequence for each of the three consecutive insertions of an hVλ gene segment with a single gene segment or an hVλ gene segment with four human Jλ gene segments The mouse homozygous for the first insertion of (Fig. 23b) was analyzed for κ and λ light chain expression in splenocytes using flow cytometry.

Briefly, spleens were harvested from groups of mice (3-7 animals per group) and crushed using glass slides. After lysing red blood cells (RBC) with ACK lysis buffer (Lonza Walkersville), splenocytes were mouse CD19 (clone 1D3; BD Biosciences), mouse CD3 (17A2; Biolegend), mouse Igκ (187.1; BD Biosciences) and mouse Igλ. It was stained with a fluorescent dye-conjugated antibody specific for (RML-42; Biolegend). Data was acquired using a BD® LSR II flow cytometer (BD Biosciences) and analyzed using FLOWJO® software (Tree Star, Inc.). ^{Table 12 shows B cells (CD19 +} ), κ light chain (CD19 ⁺ Igκ ⁺ Igλ ^_ ) and λ light chain (CD19 ⁺ Igκ ^- Igλ ⁺ ) expression observed in splenocytes from an animal group carrying each genetic modification. Represents the average% value.

In a similar experiment, mice that are homozygous for the first insertion of the 12 hVλ and 4 hJλ gene segments containing the human Vκ-Jκ genomic sequence operably linked to the mouse Cκ gene (bottom of FIG. 22B) and 40 hVλ and 1 The content of B cells in the spleen compartment from mice homozygous for the hJλ gene segment in dogs (lower part of FIG. 23B or upper part of FIG. 25B) was analyzed for Igκ and Igλ expression using a flow cytometer (described above). 26A shows Igλ and Igκ expression in ^{CD19 +} B cells for representative mice from each group. ^{The number of CD19 +} B cells per spleen was also recorded for each mouse (FIG. 26B ).

In another experiment, the B cell content of the spleen and bone marrow compartments of mouse origin that are homozygous for 40 hVλ and four hJλ gene segments comprising human Vκ-Jκ genomic sequences operably linked to the mouse Cκ gene (Fig. Bottom) was analyzed for progression through B cell development using a flow cytometer of various cell surface markers.

Briefly, two groups of mice homozygous for 40 hVλ and four hJλ gene segments containing wild-type and human Vκ-Jκ genomic sequences operably linked to a mouse Cκ gene (N=3, 9-12, respectively. Week old, male and female) were sacrificed and spleen and bone marrow harvested. Bone marrow was collected from the femur by rinsing with complete RPMI medium (fetal bovine serum, sodium pyruvate, Hepes, 2-mercaptoethanol, RPI medium supplemented with non-essential amino acids and gentamicin). RBCs from the spleen and bone marrow population were lysed with ACK lysis buffer (Lonza Walkersville) and then washed with complete RPMI medium. 1× ^{10 6} cells were incubated with anti-mouse CD16/CD32 (2.4G2, BD Biosciences) for 10 minutes on ice, and then labeled on ice with a panel of selected antibodies for 30 minutes.

Bone marrow panel: anti-mouse FITC-CD43 (1B11, BioLegend), PE-c kit (2B8, BioLegend), PeCy7-IgM (II/41, eBioscience), PerCP-Cy5.5-IgD (11-26c.2a, BioLegend) ), APC-B220 (RA3-6B2, eBioscience), APC-H7-CD19 (ID3, BD) and Pacific Blue-CD3 (17A2, BioLegend).

Bone marrow and spleen panels: anti-mouse FITC-Igκ (187.1, BD), PE-Igλ (RML-42, BioLegend), PeCy7-IgM (II/41, ebioscience), PerCP-Cy5.5-IgD (11-26c. 2a, BioLegend), Pacific Blue-CD3 (17A2, BioLegend), APC-B220 (RA3-6B2, eBioscience), APC-H7-CD19 (ID3, BD).

After staining, the cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on a FACSCANTOII® flow cytometer (BD Biosciences) and analyzed with FLOWJO® software (Tree Star, Inc.). 27A-27D show the results for the spleen compartment of one representative mouse from each group. 10A-10E show the results for the bone marrow compartment of one representative mouse from each group. 28A-28E show the results for the bone marrow compartment of one representative mouse from each group. Table 13 shows various genetic holding the deformation observed in the spleen cells of the animal group origin B cells (CD19 ^+), κ light chain ^{(CD19 + Igκ + Igλ -)} - a (Igλ ⁺ CD19 ⁺ Igκ) expression, and λ light chains Represents the average% value. ^{Table 14 shows B cells (CD19 +} ) observed in the bone marrow of mice homozygous for 40hVλ and four hJλ gene segments containing wild-type and human Vκ-Jκ genomic sequences operably linked to the mouse Cκ gene, mature B Cells (B220 ^hi IgM ⁺ ), immature B cells (B220 ^int IgM ⁺ ), immature B cells expressing κ light chain (B220 ^int IgM ⁺ Igκ ⁺ ) and immature B cells expressing λ light chain (B220 ^int IgM ⁺ Igλ ⁺ ) Represents the average% value for cells. This experiment was repeated with an additional group of mice described above and gave similar results (data not shown).

Use of the human Vλ gene in mice carrying the human λ light chain gene segment. In the third insertion of mouse and human λ sequences (hVλ5-52-hVλ3-1 and hJλ1, FIG. 23B) that are heterozygous for the first insertion of human λ sequences (hVλ3-12-hVλ3-1 and hJλ1, FIG. 23B) Mice homozygous for the human were analyzed for human λ light chain gene use by reverse transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from splenocytes.

Briefly, spleens were harvested and perfused with 10 ml of RPMI-1640 (Sigma) with 5% HI-FBS in a sterile disposable bag. Then, each bag containing a single spleen was placed in Stomacher® (Seward) and crushed in a medium setting for 30 seconds. The crushed spleen was filtered using a 0.7 μm cell strainer and then pelleted by centrifugation (1000 rpm for 10 minutes), and RBC was lysed in BD Pharm Lyse (trade name) (BD Biosciences) for 3 minutes. Splenocytes were diluted with RPMI-1640, centrifuged again, and resuspended in 1 ml of PBS PBS (Irvine Scientific). RNA was isolated from pelleted splenocytes using standard techniques known in the art.

RT-PCR was performed on splenocyte RNA using primers specific for the human hVλ gene segment and mouse Cκ gene (Table 15). The PCR product was gel-purified and cloned into a pCR2.1-TOPO TA vector (Invitrogen), and primers M13 forward orientation (GTAAAACGAC GGCCAG; SEQ ID NO: 113) and M13 reverse orientation (CAGGAAACAG) located in the vector at the position flanking the cloning site. It was sequenced using CTATGAC; SEQ ID NO: 114). 84 whole clones derived from the first and third insertions of the human λ sequence were sequenced to determine the hVλ gene usage (Table 16). The nucleotide sequence of the hVλ-hJλ1-mCκ junction for selected RT-PCR clones is shown in Figure 29.

In a similar manner, the third insertion of the human λ light chain gene sequence operably linked to the endogenous mouse Cκ gene (i.e., a 40 hVλ gene segment and four hJλ gene segments comprising the human Vκ-Jκ genomic sequence, bottom of FIG. 25B) Mice homozygous for the use of human λ light chain genes were analyzed by RT-PCR using RNA isolated from splenocytes (described above). The use of the human λ light chain gene segment for 26 selected RT-PCR clones is described in Table 17. The nucleotide sequence of the hVλ-hJλ-mCκ junction for selected RT-PCR clones is shown in FIG. 30.

In a similar manner, mice homozygous for the first insertion of a human λ light chain gene segment operably linked to the endogenous mouse Cλ2 gene (12 hVλ gene segment and hJλ1, Figs. 22A and 23A) were subjected to RNA isolated from splenocytes. The use of human λ light chain genes was analyzed by RT-PCR used (described above). The primers specific for the hVλ gene segment (Table 15) include two primers specific for the mouse Cλ2 gene; It forms a pair with either Cλ2-1 (SEQ ID NO: 162) or Cλ2-2 (SEQ ID NO: 163).

A number of hVλ gene segments rearranged to hλ1 were observed from RT-PCR clones of mouse origin carrying a human λ light chain gene segment at the endogenous mouse λ light chain locus. The nucleotide sequence of the hVλ-hJλ-mCλ2 junction for selected RT-PCR clones is shown in FIG. 31.

Figure 29 shows the sequence of the hVλ-hJλ1-mCκ junction for RT-PCR clones of mouse origin carrying the first and third insertions of the hVλ gene segment along with a single hJλ gene segment. The sequence shown in Figure 29 illustrates a unique rearrangement comprising different hVλ gene segments with hJλ1 recombined to the mouse CK gene. Heterozygous mice carrying a single modified endogenous κ site comprising a 12 hVλ gene segment and hJλ1 and homozygous mice carrying two modified endogenous κ sites comprising a 40 hVλ gene segment and hJλ1 are both mouse Cκ genes. Generating a human λ gene segment operatively linked to and producing B cells expressing the human λ light chain. These rearrangements demonstrate that the chimeric site is able to independently rearrange human λ gene segments in a number of independent B cells in these mice. Additionally, these modifications to the endogenous κ light chain locus did not render any of the hVλ gene segments inoperable, or were observed to rearrange with hJλ1, as evidenced by 16 different hVλ gene segments during B cell development. The hVλ and hJλ(Jλ1) gene segments and chimeric loci did not interfere with recombination (Table 16). Additionally, these mice produced functional antibodies comprising a rearranged human Vλ-Jλ gene segment operably linked to the mouse CK gene as part of the endogenous immunoglobulin light chain repertoire.

Figure 30 shows the sequence of the hVλ-hJλ-mCκ junction for selected RT-PCR clones from mouse homozygous for 40 hVλ and four hJλ gene segments comprising the human Vκ-Jκ genomic sequence. The sequence shown in FIG. 30 shows an additional unique rearrangement comprising a number of different hVλ gene segments, spanning the entire chimeric site, with a number of different hVλ gene segments rearranged and operably linked to the mouse CK gene. Homozygous mice comprising a modified endogenous κ site comprising 40 hVλ and four hJλ gene segments can also generate a human λ gene segment operably linked to the mouse Cκ gene and produce B cells expressing a human λ light chain. I can. These rearrangements further demonstrate that chimeric sites at all stages are able to independently rearrange human λ gene segments in a number of independent B cells in these mice. Additionally, these additional modifications to the endogenous κ light chain locus do not render each insertion of the human λ gene segment operative, or all 4 from 26 selected RT-PCR clones. It is demonstrated that the chimeric site does not interfere with recombination with the hVλ and Jλ gene segments during B cell development, as evidenced by the twelve different hVλ gene segments observed to rearrange with the canine hJλ gene segments (Table 17). Additionally, these mice produced functional antibodies comprising a human Vλ-Jλ gene segment operably linked to the mouse Cκ region as part of the endogenous immunoglobulin light chain repertoire.

Figure 31 shows the sequence of the hVλ-hJλ-mCλ2 junction for each of the three RT-PCR clones from mice homozygous for the 12 hVλ gene segment and hJλ1. The sequence shown in FIG. 31 shows an additional unique rearrangement comprising different hVλ gene segments, with hJλ1 rearranged and operably linked to the mouse Cλ2 gene (2D1 = Vλ2-8Jλ1; 2D9 = Vλ3-10Jλ1; 3E15 = Vλ3-1Jλ1). One clone demonstrated unproductive rearrangement due to the addition of N at the hVλ-hJλ junction (2D1, FIG. 31). This is common in V(D)J recombination as the linkage of gene segments appears to be inaccurate during recombination. This clone represents the non-productive recombination present in the light chain repertoire of these mice, but this means that the genetic mechanisms contributing to conjugation diversity among the antibody genes work normally in these mice to form an antibody repertoire containing light chains with greater diversity. Prove that you do.

Homozygous mice containing a 12 hVλ gene segment and a modified endogenous λ site comprising hJλ1 also generate a human λ gene segment operably linked to the endogenous mouse Cλ gene and a reverse chimera comprising an hVλ region linked to the mouse Cλ region. B cells expressing a λ light chain can be generated. These rearrangements further demonstrate that human λ light chain gene segments located at different light chain sites (i.e., λ sites) can independently rearrange human λ gene segments in multiple independent B cells in these mice. In addition, modifications to the endogenous λ light chain locus do not render insertion of the human λ gene segment inoperative either of the hVλ and/or hJλ1 gene segments, or that the chimeric site recombines the hVλ and hJλ1 gene segments during B cell development. Prove it doesn't interfere. Additionally, these mice also generated functional antibodies comprising a human Vλ-Jλ gene segment operably linked to the mouse Cλ region as part of the endogenous immunoglobulin light chain repertoire.

As shown in this example, mice bearing human λ light chain gene segments at endogenous κ and λ light chain loci are capable of rearranging the human λ light chain gene segment, and functional light chains of B cell development in both spleen and bone marrow. Because they are required at various time points, they can be expressed in the structure of the mouse Cκ and/or Cλ regions as part of the normal antibody repertoire of mice. Additionally, early subpopulations of B cells (eg, pre-, pro- and metastatic B cells) demonstrate a normal phenotype in these mice compared to wild-type litters (Figs. 27D, 28A and 28B). Small defects in the bone marrow and peripheral B cell populations are observed, which may contribute to the deletion of autoreactive immature B cells and/or suboptimal linking of the mouse heavy and human λ light chains. However, the use of Igκ/lgλ observed in these mice indicates a situation where human light chains are more expressed than those observed in mice.

Example 15. Crossing of mice expressing human λ light chain from endogenous light chain locus

Mice carrying an unrearranged human λ gene segment are crossed with another mouse containing a deletion at the opposite endogenous light chain site (κ or λ). For example, the human λ gene segment located at the endogenous κ site is the only functional light chain gene segment present in mice that also carries a deletion at the endogenous λ light chain site. In this way, the resulting offspring express only the human λ light chain as described in the previous examples. Crossing is carried out by standard art-recognized techniques and alternatively by commercial companies (eg Jackson Laboratory). Mouse strains bearing the deletion of the human λ light chain gene segment and the endogenous λ light chain site at the endogenous κ site are screened for the presence of a unique reverse chimeric (human-mouse) λ light chain and the absence of the endogenous mouse λ light chain.

Mice bearing the unrearranged human λ light chain locus also cross with mice containing the endogenous mouse heavy chain variable locus being replaced by a human heavy chain variable locus (US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®). ) Genetically engineered mice). VELOCIMMUNE® mice have a human heavy chain variable region and a mouse heavy chain constant region in response to antigenic stimulation, including those having a genome comprising a human heavy chain variable region operably linked to an endogenous mouse constant region site. It produces an antibody containing. The DNA encoding the variable region of the heavy chain of the antibody can be isolated and operably linked to the DNA encoding the human heavy chain constant region. The DNA can then be expressed in cells capable of expressing the intact human heavy chain of the antibody. In accordance with a suitable mating schedule, mice containing endogenous mouse heavy chain sites were obtained in which the endogenous κ light chain site was replaced with a human heavy chain site and an unrearranged human λ light chain site. Antibodies comprising somatically mutated human heavy chain variable regions and human λ light chain variable regions can be isolated upon immunization with an antigen of interest.

Example 16. Generation of antibodies from mice expressing human heavy chain and human λ light chain

After crossing mice containing unrearranged human λ light chain sites to various desired strains, including modifications and deletions of other endogenous Ig sites (described above), selected mice were immunized with the antigen of interest.

In general, Velocimmune (registered trademark) mice containing one of the single unrearranged human germline light chain regions are challenged with antigen and lymphocyte cells (e.g., B cells) are recovered from the animal's serum. I did. Lymphocyte cells can be fused with myeloma cell lines to produce immortal hybridoma cell lines, and these hybridoma cell lines are screened and selected to generate antibodies containing human heavy chains and human λ light chains specific for antigens used for immunization. Hybridoma cell lines were identified. DNA encoding the variable regions of the heavy and λ light chains was isolated, and the heavy and light chains were labeled and linked to the desired isotype constant regions. Due to the presence of an additional hVλ gene segment compared to the endogenous mouse λ locus, the diversity of the light chain repertoire increases rapidly and confers higher diversity to the antigen-specific repertoire upon immunization. The resulting cloned antibody sequence can subsequently be prepared in cells such as CHO cells. Alternatively, antigen specific chimeric antibodies or DNA encoding the variable domains of the light and heavy chains can be isolated directly from antigen specific lymphocytes (eg, B cells).

Initially, high affinity chimeric antibodies with human variable regions and mouse constant regions were isolated. As described above, antibodies are identified and selected for desirable properties including affinity, selectivity, epitope, and the like. The mouse constant region was replaced with the desired human constant region to generate a somatically mutated heavy chain and a human λ light chain derived from the unrearranged human λ light chain locus of the present invention. Suitable human constant regions include, for example, wild-type or modified IgG1, lgG2, IgG3, or IgG4.

Example 17. Crossing of ADAM6 mice and human λ variable mice

Any mouse described herein comprising a modification of the endogenous ADAM6 gene or an ortholog or homolog thereof, and further confers ADAM6 function to the mouse, is operatively applied to a human or mouse λ or κ constant gene. Mice were crossed with modifications containing linked human λ variable segments (eg, V and J segments). Mice containing human λ variable segments may have variable segments present at the modified endogenous λ or κ site, or in the transgene. Mice are crossed and offspring are further intercrossed as needed, offspring to fertile mice that exhibit ADAM6 function and also express human λ sequences in the structure of the human or mouse λ or κ constant region (as may be the case). Screened.

Humanized heavy chain variable locus (all or substantially all mouse V, D, and J segments replacing human V) further comprising an ectopic ADAM6 sequence (or a sequence of an ortholog or homolog of ADAM6 that confers ADAM6 function in mice). , D, and J segments) with mice comprising replacement of all or substantially all light chain V and J segments by human λ light chain V and J segments at the mouse λ site and/or the mouse κ site. I did. The offspring were further crossed as needed, and mice expressing an antibody comprising _{human V H fused with a heavy chain constant sequence and a cognate human λ V L} fused with a λ or κ light chain constant sequence were identified.

Mice were exposed to the antigen of interest and allowed to generate an immune response. An antibody specific for the antigen of interest is identified, and the human V _H sequence and the human λ variable sequence (including the human λ variable sequence linked to the mouse κ constant region) are identified and used to determine the variable domain sequence combined with the human constant region gene. Manipulated to make human antibodies.

In one example, an endogenous mouse λ comprising replacement of all or substantially all mouse heavy chain V, D, and J segments by human V, D, and J segments at the endogenous mouse heavy chain locus, and operably linked to a λ constant sequence. All or substantially all κ at the endogenous κ site by one or more human λ variable sequences comprising a light chain allele comprising replacement of all or substantially all λ light chain variable sequences by one or more human λ variable sequences at the site. Mice were generated by crossing containing a light chain allele involving replacement of the light chain variable sequence. Animals were exposed to the antigen of interest and an immune response was initiated. Antibodies that bind to the antigen of interest were found to contain human heavy chain variable domains that are cognate with human λ variable domains in the mouse λ or mouse κ constant region. Using the nucleic acid sequence encoding the variable domain, the variable sequence in combination with the human constant region sequence was engineered to create a fully human antibody.

The mice described in this example comprise one or more of the Vκ-Jκ intergenic regions described herein and in the figures.

SEQUENCE LISTING <110> REGENERON PHARMACEUTICALS, INC. <120> Humanized Light Chain Mice <130> WO/2013/096142 <140> PCT/US2012/069981 <141> 2012-12-17 <150> US 61/578,097 <151> 2011-12-20 <160> 163 <170> PatentIn version 2.0 <210> 1 <211> 754 <212> PRT <213> Mus musculus <400> 1 Met Leu Ser Leu Thr Trp Gly Met Arg Leu Val Glu Arg Pro Val Val 1 5 10 15 Pro Arg Val Leu Leu Leu Leu Phe Ala Leu Trp Leu Leu Leu Leu Val 20 25 30 Pro Val Trp Cys Ser Gln Gly His Pro Thr Trp Arg Tyr Ile Ser Ser 35 40 45 Glu Val Val Ile Pro Arg Lys Glu Ile Tyr His Thr Lys Gly Leu Gln 50 55 60 Ala Gln Arg Leu Leu Ser Tyr Ser Leu Arg Phe Arg Gly Gln Arg His 65 70 75 80 Ile Ile His Leu Arg Arg Lys Thr Leu Ile Trp Pro Arg His Leu Leu 85 90 95 Leu Thr Thr Gln Asp Asp Gln Gly Ala Leu Gln Met Glu Tyr Pro Phe 100 105 110 Phe Pro Val Asp Cys Tyr Tyr Ile Gly Tyr Leu Glu Gly Ile Leu Gln 115 120 125 Ser Met Val Thr Val Asp Thr Cys Tyr Gly Gly Leu Ser Gly Val Ile 130 135 140 Lys Leu Asp Asn Leu Thr Tyr Glu Ile Lys Pro Leu Asn Asp Ser Gln 145 150 155 160 Ser Phe Glu His Leu Val Ser Gln Ile Val Ser Glu Ser Asp Asp Thr 165 170 175 Gly Pro Met Asn Ala Trp Lys His Trp Ser His Asn Thr Gly Ser Pro 180 185 190 Ser Ser Arg Leu Glu Tyr Ala Asp Gly Ala Pro Arg Leu Ser Ser Lys 195 200 205 Asn Tyr Ala Thr His Pro Ala Ala Ile Lys Gly His Phe Gln Ala Thr 210 215 220 His Ser Val Tyr Ser Ala Ser Gly Gly Asp Lys Leu Ser Ser Thr Val 225 230 235 240 Glu Tyr Leu Phe Lys Val Ile Ser Leu Met Asp Thr Tyr Leu Thr Asn 245 250 255 Leu His Met Arg Tyr Tyr Val Phe Leu Met Thr Val Tyr Thr Glu Ala 260 265 270 Asp Pro Phe Ser Gln Asp Phe Arg Val Pro Gly Gly Gln Ala His Thr 275 280 285 Phe Tyr Glu Arg Val Phe Tyr Ala His Phe Arg Pro Asp Ala Gly Ala 290 295 300 Ile Ile Asn Lys Asn Ser Pro Gly Asp Asp Ala Val Asn Pro Ala Glu 305 310 315 320 Arg Ser Ile Cys Ser Pro Ser Ala Leu Ile Cys Leu Gly Gln His Gly 325 330 335 Arg Asn Pro Leu Phe Leu Ser Ile Ile Ile Thr Asn Arg Val Gly Arg 340 345 350 Ser Leu Gly Leu Lys His Asp Glu Gly Tyr Cys Ile Cys Gln Arg Arg 355 360 365 Asn Thr Cys Ile Met Phe Lys Asn Pro Gln Leu Thr Asp Ala Phe Ser 370 375 380 Asn Cys Ser Leu Ala Glu Ile Ser Asn Ile Leu Asn Thr Pro Asp Leu 385 390 395 400 Met Pro Cys Leu Phe Tyr Asp Arg His Val Tyr Tyr Asn Thr Ser Leu 405 410 415 Thr Tyr Lys Phe Cys Gly Asn Phe Lys Val Asp Asn Asn Glu Gln Cys 420 425 430 Asp Cys Gly Ser Gln Lys Ala Cys Tyr Ser Asp Pro Cys Cys Gly Asn 435 440 445 Asp Cys Arg Leu Thr Pro Gly Ser Ile Cys Asp Lys Glu Leu Cys Cys 450 455 460 Ala Asn Cys Thr Tyr Ser Pro Ser Gly Thr Leu Cys Arg Pro Ile Gln 465 470 475 480 Asn Ile Cys Asp Leu Pro Glu Tyr Cys Ser Gly Ser Lys Phe Ile Cys 485 490 495 Pro Asp Asp Thr Tyr Leu Gln Asp Gly Thr Pro Cys Ser Glu Glu Gly 500 505 510 Tyr Cys Tyr Lys Gly Asn Cys Thr Asp Arg Asn Ile Gln Cys Met Glu 515 520 525 Ile Phe Gly Val Ser Ala Lys Asn Ala Asn Ile Lys Cys Tyr Asp Ile 530 535 540 Asn Lys Gln Arg Phe Arg Phe Gly His Cys Thr Arg Ala Glu Glu Ser 545 550 555 560 Leu Thr Phe Asn Ala Cys Ala Asp Gln Asp Lys Leu Cys Gly Arg Leu 565 570 575 Gln Cys Thr Asn Val Thr Asn Leu Pro Phe Leu Gln Glu His Val Ser 580 585 590 Phe His Gln Ser Val Ile Ser Gly Val Thr Cys Phe Gly Leu Asp Glu 595 600 605 His Arg Gly Thr Glu Thr Ala Asp Ala Gly Leu Val Arg His Gly Thr 610 615 620 Pro Cys Ser Arg Gly Lys Phe Cys Asp Arg Gly Ala Cys Asn Gly Ser 625 630 635 640 Leu Ser Arg Leu Gly Tyr Asp Cys Thr Pro Glu Lys Cys Asn Phe Arg 645 650 655 Gly Val Cys Asn Asn Arg Arg Asn Cys His Cys His Phe Gly Trp Ser 660 665 670 Pro Pro Lys Cys Lys Glu Glu Gly His Ser Gly Ser Ile Asp Ser Gly 675 680 685 Ser Pro Pro Val Gln Arg Arg Ile Ile Lys Gln Asn Leu Glu Pro Val 690 695 700 Val Tyr Leu Arg Ile Leu Phe Gly Arg Ile Tyr Phe Leu Phe Val Ala 705 710 715 720 Leu Leu Phe Gly Ile Ala Thr Arg Val Gly Val Thr Lys Ile Phe Arg 725 730 735 Phe Glu Asp Leu Gln Ala Ala Leu Arg Ser Trp Gln Glu Gln Ala Lys 740 745 750 Asp Lys <210> 2 <211> 756 <212> PRT <213> Mus musculus <400> 2 Met Leu Ser Leu Thr Trp Gly Met Arg Leu Val Glu Arg Pro Val Val 1 5 10 15 Pro Arg Val Leu Leu Leu Leu Phe Ala Leu Trp Leu Leu Leu Leu Val 20 25 30 Pro Val Trp Cys Ser Gln Gly His Pro Thr Trp Arg Tyr Ile Ser Ser 35 40 45 Glu Val Val Ile Pro Arg Lys Glu Ile Tyr His Thr Lys Gly Leu Gln 50 55 60 Ala Gln Arg Leu Leu Ser Tyr Ser Leu His Phe Arg Gly Gln Arg His 65 70 75 80 Ile Ile His Leu Arg Arg Lys Thr Leu Ile Trp Pro Arg His Leu Leu 85 90 95 Leu Thr Thr Gln Asp Asp Gln Gly Ala Leu Gln Met Asp Tyr Pro Phe 100 105 110 Phe Pro Val Asp Cys Tyr Tyr Ile Gly Tyr Leu Glu Gly Ile Pro Gln 115 120 125 Ser Met Val Thr Val Asp Thr Cys Tyr Gly Gly Leu Ser Gly Val Met 130 135 140 Lys Leu Asp Asp Leu Thr Tyr Glu Ile Lys Pro Leu Asn Asp Ser Gln 145 150 155 160 Ser Phe Glu His Leu Val Ser Gln Ile Val Ser Glu Ser Asp Asp Thr 165 170 175 Gly Pro Met Asn Ala Trp Lys His Trp Ser His Asn Thr Gly Ser Pro 180 185 190 Ser Ser Arg Leu Glu Tyr Ala Asp Gly Ala Pro Arg Ile Ser Ser Lys 195 200 205 Asn Tyr Ala Thr His Pro Ala Ala Ile Lys Gly His Phe Gln Ala Thr 210 215 220 Asn Ser Val Tyr Asn Ser Ala Ala Gly Asp Lys Leu Ser Ser Thr Val 225 230 235 240 Gly Tyr Leu Phe Gln Val Ile Ser Leu Met Asp Thr Tyr Leu Thr Asn 245 250 255 Leu His Met Arg Tyr Tyr Val Phe Leu Met Thr Val Tyr Thr Asn Ser 260 265 270 Asp Pro Phe Arg Leu Glu Phe Ala Val Pro Gly Gly Ser Ala Tyr Asn 275 280 285 Tyr Tyr Val Ser Val Phe Tyr Asn Lys Phe Lys Pro Asp Ala Gly Val 290 295 300 Leu Leu Asn Lys Tyr Gly Pro Gln Asp Asn Gln Val Asn Pro Ala Glu 305 310 315 320 Arg Ser Ile Cys Ser Ser Leu Ala Leu Ile Cys Ile Gly Lys Tyr Asp 325 330 335 Arg Asn Pro Leu Phe Leu Ser Pro Ile Ile Thr Asn Arg Val Gly Arg 340 345 350 Ser Leu Gly Leu Lys Tyr Asp Glu Gly Tyr Cys Val Cys Gln Arg Arg 355 360 365 Asn Thr Cys Ile Met Phe Arg His Pro Gln Leu Thr Asp Ala Phe Ser 370 375 380 Asn Cys Ser Leu Ala Glu Ile Ser Asn Ile Leu Asn Thr Pro Gly Leu 385 390 395 400 Met Pro Cys Leu Phe Tyr Asp Arg His Val Tyr Tyr Asn Thr Ser Leu 405 410 415 Thr Tyr Lys Phe Cys Gly Asn Phe Lys Val Asp Asn Asp Glu Gln Cys 420 425 430 Asp Cys Gly Ser Gln Lys Ala Cys Tyr Ser Asp Pro Cys Cys Gly Asn 435 440 445 Asp Cys Arg Leu Thr Pro Gly Ser Ile Cys Asp Lys Glu Leu Cys Cys 450 455 460 Ala Asn Cys Thr Tyr Ser Pro Ser Gly Thr Leu Cys Arg Pro Ile Gln 465 470 475 480 Asn Ile Cys Asp Leu Pro Glu Tyr Cys Asn Gly Thr Lys Tyr Ile Cys 485 490 495 Pro Asp Asp Thr Tyr Leu Gln Asp Gly Thr Pro Cys Ser Glu Asp Gly 500 505 510 Tyr Cys Tyr Lys Gly Asn Cys Thr Asp Arg Asn Ile Gln Cys Met Glu 515 520 525 Ile Phe Gly Val Ser Ala Lys Asn Ala Asn Ile Lys Cys Tyr Asp Ile 530 535 540 Asn Lys Gln Arg Phe Arg Phe Gly His Cys Thr Arg Ala Glu Glu Ser 545 550 555 560 Leu Thr Phe Asn Ala Cys Ala Asp Gln Asp Lys Leu Cys Gly Arg Leu 565 570 575 Gln Cys Thr Asn Val Thr Asn Leu Pro Tyr Leu Gln Glu His Val Ser 580 585 590 Phe His Gln Ser Ile Ile Ser Gly Phe Thr Cys Phe Gly Leu Asp Glu 595 600 605 His Arg Gly Thr Glu Thr Thr Asp Ala Gly Met Val Arg His Gly Thr 610 615 620 Pro Cys Ser Lys Ser Lys Phe Cys Asp Gln Gly Ala Cys Ser Gly Ser 625 630 635 640 Leu Ser His Leu Gly Tyr Asp Cys Thr Pro Glu Lys Cys Ser Phe Arg 645 650 655 Gly Val Cys Asn Asn His Arg Asn Cys His Cys His Phe Gly Trp Lys 660 665 670 Pro Pro Glu Cys Lys Glu Glu Gly Leu Ser Gly Ser Ile Asp Ser Gly 675 680 685 Ser Pro Pro Val Gln Arg His Thr Ile Lys Gln Lys Gln Glu Pro Val 690 695 700 Val Tyr Leu Arg Ile Leu Phe Gly Arg Ile Tyr Phe Leu Phe Val Ala 705 710 715 720 Leu Leu Phe Gly Ile Ala Thr Arg Val Gly Val Thr Lys Ile Phe Arg 725 730 735 Phe Glu Asp Leu Gln Ala Thr Leu Arg Ser Gly Gln Gly Pro Ala Arg 740 745 750 Asp Lys Pro Lys 755 <210> 3 <211> 13894 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 3 gtcctaaggt agcgagggat gacagattct ctgttcagtg cactcagggt ctgcctccac 60 gagaatcacc atgccctttc tcaagactgt gttctgtgca gtgccctgtc agtggaaatc 120 tggagagcat gcttccatga gcttgtgagt agtatatcta gtaagccatg gctttgtgtt 180 aatggtgatg ttctacatac cagttctctg gcttaataat gaggtgatga ttctatgttc 240 ctgtaacgct tcctcaactg ggtcctaagt ctttcttcac tccatctatt cctctaagga 300 atgatcctga aaatcccatc acaaactata ggagatggga accatcaaaa aacacagtga 360 caaagaggtg ggaacgcatc agggttcagg aaccatattt taaaaagata tcgtaaataa 420 cttcttaaaa gagatataga caaatctcca ttaatacgga gaccagaggc ctaaggctaa 480 gaaccaatgg tggctcaagg tctcctgcta cccgaggagc aaacgtagag cagtttctaa 540 tgatttattt aaaatataga atcaaaagta ccagtttgca attttgaaag atttatttca 600 gcaatgcaac aacatcaggt ggtgccgagt ccaacacgtc ttatgtccca tgatataaac 660 aaaggccatc cagaactgtg gactggagtt ctaccttgtc ccctaatgac attcagattt 720 tttttccatt ctctttatct tagaggagac agggggctaa ctcattttac ttgtcctttg 780 cttgttcttg ccaagaacgt aaagcagctt gcaagtcttc aaacctaaat atcttagtaa 840 ctcctacacg agtggcaatg ccaaagagca gtgcaacaaa gaggaagtaa atacgaccaa 900 agagtattct taaatacact actggctcta ggttctgttt tattatgcgc ctttgaaccg 960 gaggggaccc actgtctatg ctcccactgt gtccctcttc tttgcacttt ggagggctcc 1020 aaccaaaatg gcaatggcaa ttccgacgat tgttacacac tcctctgaaa ttgcattttt 1080 ctggggtgca gtcataaccc aaacgagata aacttccatt gcaagctcct cgatcacaga 1140 acttacccct tgaacacggg gtaccatgtc tcaccaatcc agcatctgct gtttctgtcc 1200 cacgatgttc atcaagccca aagcaggtaa ccccagagat aaccgattga tggaatgaaa 1260 catgttcttg caaaaatgga agattggtga cattggtaca ctgcaacctt ccacacagct 1320 tgtcctgatc agcacaagca ttgaatgtga ggctttcttc tgctctagta caatgcccaa 1380 atcgaaaccg ttgtttgttg atgtcatagc acttaatatt agcattctta gcacttacac 1440 caaagatttc catgcattgt atgttgcgat cagtgcagtt acctttatag cagtaaccct 1500 cttctgagca tggtgtccca tcttgcagat aagtgtcatc tgggcaaatg aacttagagc 1560 cactacagta ctctggaaga tcacatatgt tctggatagg tctgcagagt gtcccagaag 1620 gactgtaagt gcaatttgca cagcataatt ctttatcaca aatgctacca ggtgttaacc 1680 tgcaatcatt tccacagcag ggatctgaat aacatgcctt ttgggagcca cagtcacact 1740 gctcattgtt atctactttg aagtttccac aaaacttata agtcaatgat gtattataat 1800 aaacatgacg gtcatagaaa agacatggca tcagatcagg agtattaagt atgttgctta 1860 tctctgcaag ggaacaattg ctgaaagcat ctgttaattg aggatttttg aacatgatgc 1920 aggtgttcct tctctggcag atacagtacc cctcatcatg ttttaggcct aaactccttc 1980 caacacgatt ggttattata atagataaaa ataaaggatt tcgaccatgt tgaccaagac 2040 aaattagggc tgagggagaa catatactcc tctcagctgg attaacagca tcatctcctg 2100 gcgaattctt gttaattata gctcctgcat caggcctaaa atgagcataa aatactctct 2160 catagaaagt atgagcctgc cctcctggaa ctcgaaaatc ttgtgaaaat ggatcagcct 2220 cggtatacac agtcatgaga aagacatagt accgcatatg aagattggtc agataggtgt 2280 ccattaaact aatgacttta aacaaatact caacagtaga tgaaagtttg tcacctccag 2340 aagcactata tacagaatgg gttgcttgaa agtggccttt tatagcagct ggatgtgtag 2400 cgtaattctt actagatagt ctgggagctc catctgcata ttccaatctg gaggagggag 2460 aacctgtatt atggctccag tgcttccatg cattcatagg ccctgtgtca tcagactcag 2520 atactatctg agaaacaagg tgttcaaagc tctgtgaatc attgaggggt ttgatttcat 2580 aggtaaggtt atccaacttt atgacccctg acaggccccc ataacaagta tccacagtga 2640 ccatggattg caggatcccc tccaggtagc caatatagta acaatctaca ggaaaaaagg 2700 ggtactccat ctgtaaggct ccttggtcat cttgagttgt cagcaacaag tgtctgggcc 2760 aaatgagtgt ctttctccgc aggtggatga tatgtctctg gccccgaaaa cgcaagctat 2820 acgagagcag tctttgtgct tgaagtcctt tggtatggta gatctccttc cgaggaataa 2880 ccacctccga tgagatgtaa cgccaagtgg gatggccttg agaacaccag actggaacca 2940 ggaggagcag ccagagtgca aatagcaaga ggaggaccct ggggaccaca ggtctttcca 3000 ctagcctcat gccccaggtc agagataaca tcctgggtgg agctaactcc ctctgctgtg 3060 gccactgcct ggtctagaaa atactgacag aggactaaaa acctcctcag gctcccaacc 3120 taagtggtta cccagacaac tggagttagg taacagtcac tgggtgtggc aggaattgag 3180 tctgaatgtg ttagctgagg ttgaggttaa atattgtcaa aagggatgtc tataaatgtg 3240 cctggacaag aaaagtcaga agcagcaagg agtgtctctg acaggctcaa tcctttcttt 3300 tctttttttg aagttcaaaa tatcatttcc acgtgaatgt atttggttcc cagtgtgact 3360 ctgggtctct ttctaggagt caatatttct ttatatcttg gctcatgttt ttcacagttg 3420 ttctaacttc ttgttttgtt ttgtttgttt gtttgtttga aagttagaag taaatactgt 3480 ctatattagc cttttagcta taaatgattg tttttatttc ttctaatcat gttttgtttg 3540 agttttggtt aaactattta caaatgagtt ttttttttcc ttttgggtgt tgctcgaaag 3600 tttggagctt tctgttaata ttgtgttgtt gtttctccaa tattattaga cctgagaatt 3660 ctacctgggt acctgtgaac tccagaattt ttaaaaattc catctcttgg gaacattatc 3720 tctgaccccg tctgaggccg aagtggctgt ccccctccaa cctttagtat ctttctttcc 3780 tgactattgg gatttcttca agcaatcagg ctgatgggtt ctcagcagtg agaccagtag 3840 actgtcggta tgaacgtcga agagtctgcc acacactccg ggttcatcaa cagtgctttc 3900 gcgtctctta cttttgtaga aggaaatgca gcctctgagt tttctccaag aaatcattga 3960 tgaaagggtg aaaagatggg tatcacccgg agttcatgac aagccctggc tcagacacgt 4020 gagcaaggtc tacagcccca aagataggct gccctgcaac atgtatttat aagataggag 4080 aaaaaaatgg gtagttggag ggttgatcaa cttacttcct ctcaaacata tatatctcat 4140 ctaagtgtgc aggggaaaac tctgtagaac tactgggata cctgctcacc cccaggagcc 4200 tcatgaataa gtctctgctt ctgccttgta gccatgagca ttactgcacc tgatacccct 4260 gcagcttcct agggaagagg gaggaagtga cttggcccct gtctggttaa ggtaagagga 4320 gataaatccc ttctcattga ttagggtgag aggggtcatg tgctctatca ttggtgaccc 4380 agttgggaca tgggtttata ccaaagtcat cactctgagg ttctgtgtac caccaggctg 4440 aactcccata tcctacatgg acataggaca acaccaagca gaaggaggtt ttaggactaa 4500 actgaaggac agagatgcgg tttctaaaca actagggagt gccagggcca gcctctctaa 4560 ccactatagg acactgtgga gtctggttac aaagagagat tactcaaggt ccttagcact 4620 gattacagag catatctcag atgccttctg ctgaccagat gtatctttgc ataatctgcc 4680 tatccagatt cagaaaattg atgccacata gccaagtgga ctttcaggaa cagacgattt 4740 aaaaacaggc agagagatgt gagagaaagg agaaggagag agagaaggga gagggagaga 4800 agagagaggg agacggagaa ggaaagaggg agaaggagaa ggagagaagg ggcatggaca 4860 gagggaggga cagaaggaga gaggagatag agagggggat aaggaagaag ggagggaggg 4920 agagagagag aaggctaagt ctttccatac ctgggtccca atacctctta taacccaagc 4980 acatggtttc acatatcaca atgcggttgg gatatagata actgtaaata cttgtgaaaa 5040 taatggggct gagatctggg gttttcatga tagtttcaaa gtcaccgtac tgactaaaac 5100 cttccactgg cccatctcca gcttcctaat ctgagggtat caaatttccc actaagtgtg 5160 tttagaaaga tctccacctt tttgcccttg tcttccagtg ccccacctac gttctggtct 5220 cccacatctg atgtcttctc agtgattctg gccctgcctg ctccacagct acaaacccct 5280 tcctataatg agctctgtgc tgagccatca tcctgaatca atccacctta agcagatgtt 5340 ttgcttattt ttcctgtgtc catactacag aggaaaggta ggcatgtaga agctgaagca 5400 tctcacctca ttccaagcac cctcagtctc taaatgtgcc cccttgtttc cagaagtgca 5460 acctcaagca tcttttattc attcatctta gagggccaca tgtgctgtag tgttataaga 5520 tgaaatttaa agcattaatt attcctaaca agccaattaa acaagccaaa aacattcatc 5580 agtcattccc atggaacctc tgaagcatct tcctgctcta accttgggtt ttccagggct 5640 gctctgggat cacaggagct gtcctgtcta ccagccatat aaaggcagac ctatcagaat 5700 tacaccagac ttctcaccat agactataaa agccagaata tcctggacag atgttataca 5760 gaaactaaga gaacacaaat gccagcccag gctactatac ccagcaaaac tctcaattac 5820 catcgatgaa gaaaccaaga tattccatta caagtccaaa tttacacaat atctttccat 5880 aaatccagcc ctacaaagga tagcagatgg aaaactccaa cacaggtagg aaaactacac 5940 cctagaaaga gcactaaagt aatcatcttt caacacactc aaaagaagat aaccacacaa 6000 acataattcc acctctaaca acaaaaataa agtaggcaac aatcactatt ccttaatatc 6060 tcttttaaca tcaatggact caattctcca ataaaaagac atagactaac agactgaata 6120 cataaacagg acacagcatt ttgctgcata aagcaaacac agcgttactt ttttttttct 6180 aaatgacatt ttttattaga tattgtcttt attgacattt caaatgttat cccctttcct 6240 ggtttaccct ctgaaatccc ctatctcctc cccctccccc tgctcaccaa tccacccact 6300 cccacttcca ggccctggca atcccctata tttgggcata gagccttcac aggaccaagg 6360 tactctcctt gcattgatga ccaactagtc cattctctgc tacaaatgca gctagatcta 6420 tgagtcccac catgttttct tttgttggtg gtttcatgcc agggagctct tggagtactg 6480 attggttcat attgttgttc tccctatggg gttacaaaac ccttcaactt cttgggtcct 6540 ttctctggct gcctcattgg ggaccttgtg cgaagtccaa tggatgactg tgagcatcca 6600 cttctgtatt tgccaggcac tggcagagcc tctcagaaga cagctatatc aagatcctgg 6660 cagcaagctc ttgttggtat ccacaaaagt gtctggtggt tgtctatggg atggatcccc 6720 aaaggggcag tctctggatg gtcattcctt cagtctctgt tccacacttt gtctctttaa 6780 ctccttccat gactatttta ttcctccctc taagaaggac cgaagtattc atactttggt 6840 cttccttctt gaaattcatg tgttttgtga attgtatctt tgatattccg aacttctggg 6900 ctaatatcca cttatcagtg agtgaatatc atgtgtgttc ttatgtgatt gagttacctc 6960 actcaggatg atatcctcca gaaccatcca tttgtctaag aatttaatga attcattgtt 7020 tttaatagct gaggagtact ccattgtgta aatgtaccac attttctgta cccattgttc 7080 tcttgaggga catctgggtt ctttaaagct tctggacatt aaatataagg ctgctatgga 7140 aatagtggag aatgtgtcct tattacatgt tggagcatct tctgggtata tgcccaggag 7200 tgctattgct ggatcctctg atagtactat gtccaatttt ctgaggaact gccaaactga 7260 tttacagagt ggttgtacca gcttgcaatt ccaccagcaa tggagaaatg ttccccttcc 7320 tccacatcct caccaacatc tgctgtcacc tcaatttgtt cttagtgatt cagacaggtg 7380 tgaggtggaa tatcagggtt gtttggcatt tccctgatga ctagtgatat tgaaaaaaat 7440 tttaagtgtt tctcagccat tcagtattct tcagttgaga attcactgtt tagctctgta 7500 ctcaggtttt tttaataggg ttatttggtt ttctggagtc taacgtcttg aattctttct 7560 atatattgga tattagccct ctgtcatatt taggattggt aaagatcttt cccaatatgt 7620 tggctgcctt tttgtgtcct ttgccttaca gaaccttttt aattttatga ggtcccattt 7680 gctaattctt cattttacag cacaagccat tggtgttctg ttcaaaaatc tttccccctg 7740 aaccctatct tcgaggatct tccccacttt ctcctctata agtttcagtg tctctattat 7800 tgtgctgagg ggtaccgaag ttcctattcc gaagttccta ttctctagaa agtataggaa 7860 cttccctagg gtttaaaccc gcggtggagc tctgatgtgg gaacgcttca gtgttcagga 7920 accatatgat ttatttaaaa tatagaatca aaagtaccaa tttgcagttt tgaaagattt 7980 attccagtgt aagcattagc aatgcaccaa catcaggtga tttctgaatc caacacgtct 8040 tatgtcctca tgatattaaa aaaaaaaaaa ggccatccag aactgtgaac ttgagttcta 8100 ccttgttccc tactgacatt cagattttct tttttgcatt ctctttatct tacaggagac 8160 aggaggggag ggctaactca ttttactttg gcttgtccct tgctggtcct tgcccagaac 8220 gtaaagtagc ttgcaagtct tcaaatctaa aaatcttagt aactcctaca cgagtggcaa 8280 tgccaaagag cagtgcaaca aagaggaagt aaatacgacc aaagagtatt cttaaataca 8340 ccactggctc ttgtttttgt tttattgtgt gcctttgaac tggaggggac ccactgtcta 8400 tgctcccact tagtccctct tctttgcact ctggaggctt ccaaccaaaa tgacaatggc 8460 aattccgatg attgttacac actcctctaa aactgcattt ttctggggtg cagtcataac 8520 ccaaatgaga taaacttcca ctgcaagctc cttgatcaca gaacttactt ttggagcagg 8580 gggtaccatg tctcaccatt ccagcatctg ttgtttctgt cccacgatgt tcatcaagcc 8640 caaagcaggt aaacccagag ataatcgatt gatggaatga aacatgttct tgcaaatatg 8700 gaagattggt gacattggta cactgcaacc ttccacacag cttgtcctga tcagcacaag 8760 cattgaatgt gaggctttct tctgctctag tacaatgccc aaatcgaaac cgttgtttgt 8820 tgatgtcata gcacttaata ttagcattct tagcacttac accaaagatt tccatgcatt 8880 gtatgttgcg atcagtgcag ttacctttat agcagtaacc atcttctgag catggtgtcc 8940 catcttgcag ataagtgtca tctgggcaaa tgtatttagt cccattacag tactctggaa 9000 gatcacatat gttctggata ggtctgcaga gtgtcccaga aggactgtaa gtgcaatttg 9060 cacagcataa ttctttatca caaatgctac caggtgttaa cctgcaatca tttccacagc 9120 agggatctga ataacatgcc ttttgggagc cacagtcaca ctgctcatcg ttatctactt 9180 tgaagtttcc acaaaactta taagtcaatg atgtattata ataaacatga cggtcataga 9240 aaagacatgg catcagacca ggagtattaa gtatgttgct tatctctgca agggaacaat 9300 tgctgaaagc atctgttaat tgaggatgtc tgaacataat gcaggtgttc cttctctggc 9360 agacacagta cccctcatca tattttaagc ctaaactcct tccaacacga ttggttatta 9420 taggagataa aaataaagga tttcgatcat atttaccaat acaaattagg gctaaggaag 9480 aacatatact cctctcagct ggattaacct ggttatcttg tggcccatac ttattaagta 9540 aaactcctgc atcaggctta aatttattat aaaagactga cacatagtaa ttataagccg 9600 accctcctgg aactgcaaac tcaagtcgaa atggatcaga attggtgtac acagtcatga 9660 gaaagacata gtaccgcata tgaagattgg tcagataggt gtccattaaa ctaatgactt 9720 gaaacaaata cccaacagta gatgaaagtt tgtcacctgc agcagaatta tatacagaat 9780 tggttgcttg aaagtggcct tttatagcag ctggatgtgt agcgtagttc ttactagata 9840 ttctgggagc tccatctgca tattccaatc tggaggaggg agaacctgta ttatggctcc 9900 agtgcttcca tgcattcata ggccctgtgt catcagactc agatactatc tgagaaacaa 9960 ggtgttcaaa gctctgtgaa tcattgaggg gtttgatttc ataggtaagg tcatctaact 10020 tcatgacccc tgacaggccc ccataacaag tatccacagt gaccatggat tgtgggatcc 10080 cctccaggta gccaatatag taacaatcta caggaaaaaa ggggtaatcc atctgtaagg 10140 ctccttggtc atcttgagtt gtcagcaaca agtgtctggg ccaaatgagt gtctttctcc 10200 gcaggtggat gatatgtctc tggccccgaa aatgcaagct atatgagagc agtctttgtg 10260 cttgaagtcc tttggtatgg tagatctcct tccgaggaat aaccacctcc gatgagatgt 10320 aacgccaagt aggatggcct tgagaacacc agactggaac caggaggagc agccagagtg 10380 caaatagcaa gaggaggacc ctggggacca caggtctttc cactagcctc atgccccagg 10440 tcagagataa catcctgggt ggagctaaat ccctctgctg tggccactgc ctggtctaga 10500 aaatactgac agaggactaa aaacctcctc aggctcccaa cctaagtggt tacccagaca 10560 actggagtta ggtaacagtc actgggtgtg gcaggaattg agtctgaatg tgttagctga 10620 ggttgaggtt aaatattgtc aaaagggatg tctataaatg tgcctggaca agaaaagtca 10680 gaagcagcaa ggagtgtctc tgacaggctc aatcctttct tttctttttt tgaagttcaa 10740 aatatcattt ccacgtgaat gtatttggtt cccagtgtga ctctgggtct ctttctagga 10800 gtcaatattt ctttatatct tggctcatgt ttctcacagt tgttctaatt tcttgttttg 10860 ttttgtttgt ttgtttgaac gttagtagta aatactgtct atattagcct tttagctata 10920 aatgattgtt tttatttctt ctaatcatat tttgtttgag ttttggttaa actatttaca 10980 aatgagtttt ttttttttcc ttttgggtgt tgctcgaaag tttggagctt tctgttaata 11040 ttgtgttgtt atttttccaa tattattaga cctgagaatt ctatctgggt acctgtgaac 11100 tctagaattt ttaaaaattc catctcttgg gaacattacc tctgaccccg tctgaggccg 11160 aagtggctgt ccccctccaa cctttagtat ctttctttcc tgactattgg gatttcttca 11220 agcaatcagg ctgatgggtt ctcagcagtg agaccagtag actgccggta tgaacgtcga 11280 agagactgcc acacactcca ggttcatcaa cagtgctttc gcgtctctta cttttgtaga 11340 aggaaaagca gcctctgagt tatctccaag aaatcattaa tgaaagagtt aaaagatggg 11400 tatcacccgg agttcatgac aagccctggc tcagacacgt gagcaaggtc tacagcccca 11460 aagataggct gccctgcaac atgtatttat aagatagaag aaaaaaatgg gtggttggag 11520 ggttgatcaa cttacttcct ctcaaacata tatatctcat ctaagtgtgc aggggaaaac 11580 tctgtaggac tactgggatt gttattatca ttattattat tattattatt attattatta 11640 ttattattat tattaactta aggcatttta ttagatattt tcttcattta gttttcaaat 11700 gttatccccg gaacctccta tactctctcc ctgccctgct ccccaaccca cccactccta 11760 catcctggcc ctggcattcc cctatactgt ggcagatgat cttcgtaaga ccaagagcct 11820 ttcctcccat tgatggccta ctaggctatc ctcttttaca tatgcaacta gagtcacagc 11880 tctggggagg tattgcttag ttcatattgt ttttcctcct atagggttgc agatcccttt 11940 agctccttgg gtactttctc tagctcctcc attgggggcc ctgtgttcca tccaatagat 12000 gactgtgagc atccacttct gtatttgcca ggtattggca tggatcttac tgcaccttct 12060 gaactctcta agcagctttc ctggtcacct ccaggagcct catgaataag tctctgcttc 12120 ccccttgtgg ctatgagcat tactgcacct gatacaccct gcagcttcct agggaagagg 12180 gaggaagtgg cttggcccct gtctggttaa ggtaagagga gataaatccc ttctcatgaa 12240 ttagggtgag aagggtcatg tgctctatca ttggtgacca acttggggac atgggcttat 12300 acagtcatca ctctgaggct ctgtgtacca ccagactgaa ctcccatatc ctacatgcac 12360 ataggacaac accaagtaga aggaggtttt aggactaaac tgaaggacag agatggggtt 12420 tctaaacaac tagggagtgc cagggccagc ctctctaacc actataggac actatggagt 12480 ctggttacaa agagagatta ctcaaggtcc ttagcactga ttacagagca tatctcagat 12540 gccttctgct gaccagatgt atctttgcat aatctgccta tccagattca gaaaattgat 12600 gccacatagc caagtggact ttcaggaaca gacgatttaa aaacaggcag agagatgtga 12660 gagaaaggag aaggagagag agaagggaga gggagagaag agagagggag acggagaagg 12720 aaagagggag aaggagaagg agagaagggg catggacaga gggagggaca gaaggagaga 12780 ggagatagag agggggataa ggaagaaagg agggagggag agagagagaa ggctaagtct 12840 ttccatacct gggtcccaat acctcttata acccaagcac atggtttcag atatcacaat 12900 gcggttggga tatagataac tgtaaatact tgtgaaaata atggggctga gatctggggt 12960 tttcatgata gtttcaaagt cactgtactg actaaaacct tccactggcc catctccagc 13020 ttgttaatct gagggtatca aatttcccac taagtgtgtt tagaaagatc tccacctttt 13080 tgccctagtc ttccagtgcc ccacctacgt tctggtctcc cacatctgat gtcttctcag 13140 tgattctggc cctgcctgct ccacagctac aaaccccttc ctataatgag ctctgtgctg 13200 agccatcatc ctgaatcaat ccaccttaag cagatgtttt gcttattttt cctgtgtcca 13260 tactacagag gaagggtagg catgtagaag ctgaggcatc tcatctcact ctaagcaccc 13320 tcagtctcta aatgtgcccc tttgtttcca gcagttcagc ctcaagcatc ttttattcac 13380 tcgtcttaga gggacacatg tgctgtagtg ttataagatg aaatttaaag cattagttat 13440 tcccaacaag ccaattaaac aagccaaaaa cattcatcag tcattcccat ggaacctctg 13500 aagcatcttc ctgctctaac cttgagtttc ctagggctgc tgtgggatca caggagctgt 13560 cctgtttacc agcctatcct gtcccacggg attcagttat tagtgggtgc gagggggacc 13620 gcaaacctgg aagaaaatgg gattggaaga gaaaagagaa acgaagacca agtagatctt 13680 ttcctatcaa ggtcttcgtt tattaggctg aggtgcctgg tgtaaagcat gcatcgcggg 13740 gaataggaag gggtcgaggg ggaattttac aaagaacaaa gaagcgggca tctgctgaca 13800 tgagggccga agtcaggctc caggcagcgg gagctccacc gcggtggcgc catttcatta 13860 cctctttctc cgcacccgac atagataaag ctta 13894 <210> 4 <211> 251 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 4 ccagcttcat tagtaatcgt tcatctgtgg taaaaaggca ggatttgaag cgatggaaga 60 tgggagtacg gggcgttgga agacaaagtg ccacacagcg cagccttcgt ctagaccccc 120 gggctaacta taacggtcct aaggtagcga ggggatgaca gattctctgt tcagtgcact 180 cagggtctgc ctccacgaga atcaccatgc cctttctcaa gactgtgttc tgtgcagtgc 240 cctgtcagtg g 251 <210> 5 <211> 245 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 5 aggggtcgag ggggaatttt acaaagaaca aagaagcggg catctgctga catgagggcc 60 gaagtcaggc tccaggcagc gggagctcca ccgcggtggc gccatttcat tacctctttc 120 tccgcacccg acatagataa agcttatccc ccaccaagca aatcccccta cctggggccg 180 agcttcccgt atgtgggaaa atgaatccct gaggtcgatt gctgcatgca atgaaattca 240 actag 245 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 6 caggtacagc tgcagcagtc a 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 7 ggagatggca caggtgagtg a 21 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 8 tccaggactg gtgaagc 17 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 9 tagtcccagt gatgagaaag agat 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 10 gagaacacag aagtggatga gatc 24 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 11 tgagtccagt ccaggga 17 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 12 aaaaattgag tgtgaatgga taagagtg 28 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 13 aaccctggtc agaaactgcc a 21 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 14 agagaaacag tggatacgt 19 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 15 aactacgcac agaagttcca gg 22 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 16 gctcgtggat ttgtccgc 18 <210> 17 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 17 cagagtcacg attacc 16 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 18 tgagcagcac cctcacgtt 19 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 19 gtggcctcac aggtatagct gtt 23 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 20 accaaggacg agtatgaa 18 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 21 gctagtagtg gggcctacag gccttttgat atc 33 <210> 22 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 22 gcaaaagccc aggggagtgg gagctactac acctatgctt ttgatatc 48 <210> 23 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 23 gcgagagagg gtatagtggg aactactgag gactttgatt ac 42 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 24 gcgagaggga cagtgggagc cctctttgac tac 33 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 25 gcgaaaccta gtgggagcta ctcctggttc gacccc 36 <210> 26 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 26 gcgagaggag gagggtataa ctggaactcg aatgcttttg atatc 45 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 27 gcgagaggat ataactggaa ctactttgac tac 33 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 28 gcgaaagagt ataactggaa ccactggtac tttgactac 39 <210> 29 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 29 gcgagagaga taactggaac cccctttgac tac 33 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 30 gcgaggggat ataactggaa cttttctttt tttgactac 39 <210> 31 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 31 gcgagaggta actggaactc tctgggcttt gactac 36 <210> 32 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 32 gcgaaaaggg ctactatggt tcggggagct cttgactac 39 <210> 33 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 33 gcgagagata ttactatggt tcggggagtt attataacga aggtctacgg tatggacgtc 60 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 34 gcgagagagt atagcagctt tgactac 27 <210> 35 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 35 gcgagagaga gtatagcagc tcgttgtgac tac 33 <210> 36 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 36 gcaagagagg ataggagctc gcccctcggg tactttgact ac 42 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 37 gcgagagatc ttggggaagg ctac 24 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 38 accacccata actggggagg gtttgactac 30 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 39 gcgagagata ggggaccg 18 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 40 caacagagtt atagtacccc tccggagacg 30 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 41 caacagctta atagttaccc tcggacg 27 <210> 42 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 42 caacagctta atagttacca ttcact 26 <210> 43 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 43 caacatttta atagttaccc gctcact 27 <210> 44 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 44 cagcagtata ataactggcc tctcact 27 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 45 ctacagcata atagttaccc gtggacg 27 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 46 ctacagcata atagttaccc tcggacg 27 <210> 47 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 47 cagcagtatg gtagctcacc tcggacg 27 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 48 atgcaaggta cacactggcc gtggacg 27 <210> 49 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 49 atgcaaggtt cacactggcc gtacact 27 <210> 50 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 50 atgcaaggta cacactggcc gctcact 27 <210> 51 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 51 caacagtatg ataatctccc tcccact 27 <210> 52 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 52 caacagtatg ataatctccc attcact 27 <210> 53 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 53 caacagtatg ataatctccc cgtcact 27 <210> 54 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 54 caacagtatg ataatctccc gatcacc 27 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 55 caacggattt acaatgccga cacc 24 <210> 56 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 56 caacagagtt acagtacccc catgtacact 30 <210> 57 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 57 caacagagtt acagtacccc tctcact 27 <210> 58 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 58 caacagagtt acagtactcc tcccact 27 <210> 59 <211> 219 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 59 actttcagaa tgttcttgaa cagtctctga gaaacacgga agacggccgc ataacttcgt 60 atagtataca ttatacgaag ttattctaga cccccgggct cgataactat aacggtccta 120 aggtagcgac tcgagataac ttcgtataat gtatgctata cgaagttatc catggtaagc 180 ttacgtggca tacagtgtca gattttctgt ttatcaagc 219 <210> 60 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 60 agctgaatgg aaacaaggca a 21 <210> 61 <211> 19 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 61 ggagacaatg ccccagtga 19 <210> 62 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 62 tcccataggg ctaggatttc c 21 <210> 63 <211> 19 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 63 tcccctcaca ctgttcccc 19 <210> 64 <211> 19 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 64 ggtggagagg ctattcggc 19 <210> 65 <211> 17 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 65 gaacacggcg gcatcag 17 <210> 66 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 66 tcaacctttc ccagcctgtc t 21 <210> 67 <211> 24 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 67 ccccagagag agaaaacaga tttt 24 <210> 68 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 68 ccctggtgaa gcatgtttgc 20 <210> 69 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 69 tgtggcctgt ctgccttacg 20 <210> 70 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 70 cacacctaga ccccggaagt c 21 <210> 71 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 71 tcgctttgcc agttgattct c 21 <210> 72 <211> 17 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 72 tgcggccgat cttagcc 17 <210> 73 <211> 18 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 73 ttgaccgatt ccttgcgg 18 <210> 74 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 74 gcaaacaaaa accactggcc 20 <210> 75 <211> 19 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 75 ggccacattc catgggttc 19 <210> 76 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 76 ccatgactgg gcctctgtag ac 22 <210> 77 <211> 25 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 77 caagtcaggg tgctaatgct gtatc 25 <210> 78 <211> 19 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 78 cacagcttgt gcagcctcc 19 <210> 79 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 79 gggcactgga tacgatgtat gg 22 <210> 80 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 80 tcataggtag gtctcagttt g 21 <210> 81 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 81 tgatctgcgc tgtttcatcc t 21 <210> 82 <211> 31 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 82 tgacatgaac catctgtttc tctctcgaca a 31 <210> 83 <211> 29 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 83 agagacgctc cgaggtcaag gtgctctag 29 <210> 84 <211> 23 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 84 tgggcacaac agacaatcgg ctg 23 <210> 85 <211> 16 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 85 accctctgct gtccct 16 <210> 86 <211> 26 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 86 ccaagcagga ggtgctcagt tcccaa 26 <210> 87 <211> 24 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 87 tccacactgt cggctgggag ctca 24 <210> 88 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 88 acgagcgggt tcggcccatt c 21 <210> 89 <211> 37 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 89 ctgttcctct aaaactggac tccacagtaa atggaaa 37 <210> 90 <211> 27 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 90 tgccgcttat acaacactgc catctgc 27 <210> 91 <211> 37 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 91 agaagaagcc tgtactacag catccgtttt acagtca 37 <210> 92 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 92 gggctacttg aggaccttgc t 21 <210> 93 <211> 23 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 93 gacagccctt acagagtttg gaa 23 <210> 94 <211> 23 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 94 aagaccagga gctctgccta agt 23 <210> 95 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 95 cccatcacga actgaagttg ag 22 <210> 96 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 96 cagggcctcc atcccaggca 20 <210> 97 <211> 28 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 97 ccccagtgtg tgaatcactc taccctcc 28 <210> 98 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 98 cctctcctcc tcaccctcct 20 <210> 99 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <220> <221> variation <222> (4)...(4) <223> r=a or g <220> <221> variation <222> (9)...(9) <223> s=c or g <220> <221> variation <222> 11, 12, 13 <223> y=c or t <400> 99 atgrccdgst yyyctctcct 20 <210> 100 <211> 18 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 100 ctcctcactc agggcaca 18 <210> 101 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <220> <221> variation <222> (18)...(18) <223> s=c or g <400> 101 atggcctggg ctctgctsct 20 <210> 102 <211> 19 <212> DNA <213> artificial sequence <220> <223> synthetic <220> <221> variation <222> (11)...(11) <223> y=c or t <220> <221> variation <222> (13)...(13) <223> s=c or g <400> 102 atggcctgga ycsctctcc 19 <210> 103 <211> 23 <212> DNA <213> artificial sequence <220> <223> synthetic <220> <221> variation <222> 11, 16, 18, 21 <223> y=c or t <220> <221> variation <222> (15)...(15) <223> r=a or g <220> <221> variation <222> (20)...(20) <223> m=a or c <400> 103 tcaccatggc ytggrycycm ytc 23 <210> 104 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 104 tcaccatggc ctgggtctcc tt 22 <210> 105 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <220> <221> variation <222> (16)...(16) <223> m=a or c <220> <221> variation <222> (19)...(19) <223> y=c or t <400> 105 tcaccatggc ctggamtcyt ct 22 <210> 106 <211> 26 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 106 tcaccatggc ctgggctcca ctactt 26 <210> 107 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 107 tcaccatggc ctggactcct 20 <210> 108 <211> 23 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 108 tcaccatggc ctggatgatg ctt 23 <210> 109 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 109 taaatatggc ctgggctcct ct 22 <210> 110 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 110 tcaccatgcc ctgggctctg ct 22 <210> 111 <211> 22 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 111 tcaccatggc cctgactcct ct 22 <210> 112 <211> 30 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 112 cccaagctta ctggatggtg ggaagatgga 30 <210> 113 <211> 16 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 113 gtaaaacgac ggccag 16 <210> 114 <211> 17 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 114 caggaaacag ctatgac 17 <210> 115 <211> 440 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 115 gggcctgggc tctgctgctc ctcaccctcc tcactcaggg cacagggtcc tgggcccagt 60 ctgccctgac tcagcctccc tccgcgtccg ggtctcctgg acagtcagtc accatctcct 120 gcactggaac cagcagtgac gttggtggtt ataactatgt ctcctggtac caacagcacc 180 caggcaaagc ccccaaactc atgatttatg aggtcagtaa gcggccctca ggggtccctg 240 atcgcttctc tggctccaag tctggcaaca cggcctccct gaccgtctct gggctccagg 300 ctgaggatga ggctgattat tactgcagct catatgcagg cagcaacaat ttcgtcttcg 360 gaactgggac caaggtcacc gtcctagggg ctgatgctgc accaactgta tccatcttcc 420 caccatccag taagcttggg 440 <210> 116 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 116 atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcacta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtaagcttgg g 441 <210> 117 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 117 atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcagta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtaagcttgg g 441 <210> 118 <211> 438 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 118 atggcctggg ctctgctcct caccctcctc actcagggca cagggtcctg ggcccagtct 60 gccctgactc agcctccctc cgcgtccggg tctcctggac agtcagtcac catctcctgc 120 actggaacca gcagtgacgt tggtggttat aactatgtct cctggtacca acagcaccca 180 ggcaaagccc ccaaactcat gatttatgag gtcagtaagc ggccctcagg ggtccctgat 240 cgcttctctg gctccaagtc tggcaacacg gcctccctga ccgtctctgg gctccaggct 300 gaggatgagg ctgattatta ctgcagctca tatgcaggca gcaacaatta tgtcttcgga 360 actgggacca aggtcaccgt cctaggggct gatgctgcac caactgtatc catcttccca 420 ccatccagta agcttggg 438 <210> 119 <211> 438 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 119 atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcagta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa tgtcttcgga 360 actgggacca aggtcaccgt cctaggggct gatgctgcac caactgtatc catcttccca 420 ccatccagta agcttggg 438 <210> 120 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 120 atggcctggg ctctgctcct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcagta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtaagcttgg g 441 <210> 121 <211> 442 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 121 atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcccag 60 tctgccctga ctcagcctcc ctccgcgtcc gggtctcctg gacagtcagt caccatctcc 120 tgcactggaa ccagcagtga cgttggtggt tataactatg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gaggtcagta agcggccctc aggggtccct 240 gatcgcttct ctggctccaa gtctggcaac acggcctccc tgaccgtctc tgggctccag 300 gctgaggatg aggctgatta ttactgcagc tcatatgcag gcagcaacaa tttatgtctt 360 cggaactggg accaaggtca ccgtcctagg ggctgatgct gcaccaactg tatccatctt 420 cccaccatcc agtaagcttg gg 442 <210> 122 <211> 428 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 122 ccttcatttt ctccacaggt ctctgtgctc tgcctgtgct gactcagccc ccgtctgcat 60 ctgccttgct gggagcctcg atcaagctca cctgcaccct aagcagtgag cacagcacct 120 acaccatcga atggtatcaa cagagaccag ggaggtcccc ccagtatata atgaaggtta 180 agagtgatgg cagccacagc aagggggacg ggatccccga tcgcttcatg ggctccagtt 240 ctggggctga ccgctacctc accttctcca acctccagtc tgacgatgag gctgagtatc 300 actgtggaga gagccacacg attgatggcc aagtcggttg tgtcttcgga actgggacca 360 aggtcaccgt cctaggggct gatgctgcac caactgtatc catcttccca ccatccagta 420 agcttggg 428 <210> 123 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 123 atgacctgct cccctctcct cctcaccctt ctcattcact gcacagggtc ctgggcccag 60 tctgtgttga cgcagccgcc ctcagtgtct gcggccccag gacagaaggt caccatctcc 120 tgctctggaa gcagctccaa cattgggaat aattatgtat cctggtacca gcagctccca 180 ggaacagccc ccaaactcct catttatgac aataataagc gaccctcagg gattcctgac 240 cgattctctg gctccaagtc tggcacgtca gccaccctgg gcatcaccgg actccagact 300 ggggacgagg ccgattatta ctgcggaaca tgggatagca gcctgagtgc ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtgagcagtt a 441 <210> 124 <211> 441 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 124 atgacctgct cccctctcct cctcaccctt ctcattcact gcacagggtc ctgggcccag 60 tctgtgttga cgcagccgcc ctcagtgtct gcggccccag gacagaaggt caccatctcc 120 tgctctggaa gcagctccaa cattgggaat aattatgtat cctggtacca gcagctccca 180 ggaacagccc ccaaactcct catttatgac aataataagc gaccctcagg gattcctgac 240 cgattctctg gctccaagtc tggcacgtca gccaccctgg gcatcaccgg actccagact 300 ggggacgagg ccgattatta ctgcggaaca tgggatagca gcctgagtgc ggcttttttt 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtgagcagtt a 441 <210> 125 <211> 345 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 125 cccgggcaga gggtcaccat ctcttgttct ggaagcagct ccaacatcgg aagtaatact 60 gtaaactggt accagcagct cccaggaacg gcccccaaac tcctcatcta tagtaataat 120 cagcggccct caggggtccc tgaccgattc tctggctcca agtctggcac ctcagcctcc 180 ctggccatca gtgggctcca gtctgaggat gaggctgatt attactgtgc agcatgggat 240 gacagcctga atggttatgt cttcggaact gggaccaagg tcaccgtcct aggggctgat 300 gctgcaccaa ctgtatccat cttcccacca tccagtgagc agtta 345 <210> 126 <211> 432 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 126 atggcctgga cccctctcct gctccccctc ctcactttct gcacagtctc tgaggcctcc 60 tatgagctga cacagccacc ctcggtgtca gtgtccccag gacaaacggc caggatcacc 120 tgctctggag atgcattgcc aaaaaaatat gcttattggt accagcagaa gtcaggccag 180 gcccctgtgc tggtcatcta tgaggacagc aaacgaccct ccgggatccc tgagagattc 240 tctggctcca gctcagggac aatggccacc ttgactatca gtggggccca ggtggaggat 300 gaagctgact actactgtta ctcaacagac tacagtggta atcatgtctt cggaactggg 360 accaaggtca ccgtcctagg ggctgatgct gcaccaactg tatccatctt cccaccatcc 420 agtgagcagt ta 432 <210> 127 <211> 426 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 127 atggcctgga ctcctctctt tctgttcctc ctcacttgct gcccagggtc caattcccag 60 gctgtggtga ctcaggagcc ctcactgact gtgtccccag gagggacagt cactctcacc 120 tgtggctcca gcactggagc tgtcaccagt ggtcattatc cctactggtt ccagcagaag 180 cctggccaag cccccaggac actgatttat gatacaagca acaaacactc ctggacacct 240 gcccggttct caggctccct ccttgggggc aaagctgccc tgaccctttc gggtgcgcag 300 cctgaggatg aggctgagta ttactgcttg ctctcctata gtggtgctta tgtcttcgga 360 actgggacca aggtcaccgt cctaggggct gatgctgcac caactgtatc catcttccca 420 ccatcc 426 <210> 128 <211> 331 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 128 agtggtcctg ggacagacgg ccaggattac ctgtggggga aacaacattg gaagtaaaaa 60 tgtgcactgg taccagcaga agccaggcca ggcccctgtg ctggtcatct atagggataa 120 caaccggccc tctgggatcc ctgagcgatt ctctggctcc aactcgggga acacggccac 180 cctgaccatc agcagagccc aagccgggga tgaggctgac tattactgtc aggtgtggga 240 cagcagcact tatgtcttcg gaactgggac caaggtcacc gtcctagggg ctgatgctgc 300 accaactgta tccatcttcc caccatccag t 331 <210> 129 <211> 417 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 129 actcctctcc tcctcctgtt cctctctcac tgcacaggtt ccctctcgca ggctgtgctg 60 actcagccgt cttccctctc tgcatctcct ggagcatcag ccagtctcac ctgcaccttg 120 cgcagtggca tcaatgttgg tacctacagg atatactggt accagcagaa gccagggagt 180 cctccccagt atctcctgag gtacaaatca gactcagata agcagcaggg ctctggagtc 240 cccagccgct tctctggatc caaagatgct tcggccaatg cagggatttt actcatctct 300 gggctccagt ctgaggatga ggctgactat tactgtatga tttggcacag cagcgcttat 360 gtcttcggaa ctgggaccaa ggtcaccgtc ctaggggctg atgctgcacc aactgta 417 <210> 130 <211> 393 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 130 tttctgttcc tcctcacttg ctgcccaggg tccaattctc agactgtggt gactcaggag 60 ccctcactga ctgtgtcccc aggagggaca gtcactctca cctgtgcttc cagcactgga 120 gcagtcacca gtggttacta tccaaactgg ttccagcaga aacctggaca agcacccagg 180 gcactgattt atagtacaag caacaaacgc tcctggaccc ctgcccggtt ctcaggctcc 240 ctccttgggg gcaaagctgc cctgacactg tcaggtgtgc agcctgagga cgaggctgag 300 tattactgcc tgctctacta tggtggtgct tatgtcttcg gaactgggac caaggtcacc 360 gtcctagggg ctgatgctgc accaactgta tcc 393 <210> 131 <211> 417 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 131 atggcctggg ctctgctgct cctcactctc ctcactcagg acacagggtc ctgggcccag 60 tctgccctga ctcagcctgc ctccgtgtct gggtctcctg gacagtcgat caccatctcc 120 tgcactggaa ccagcagtga tgttgggagt tataaccttg tctcctggta ccaacagcac 180 ccaggcaaag cccccaaact catgatttat gagggcagta agcggccctc aggggtttct 240 aatcgcttct ctggctccaa gtctggcaac acggcctccc tgacaatctc tgggctccag 300 gctgaggacg aggctgatta ttactgctgc tcatatgcag gtagtagcac ttatgtcttc 360 ggaactggga ccaaggtcac cgtcctaggg gctgatgctg caccaactgt atccatc 417 <210> 132 <211> 348 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 132 cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacag 120 cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180 tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cacttatgtc 300 ttcggaactg ggaccaaggt caccggcctg ggggctgatg ctgcacca 348 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 133 aacaaccgag ctccaggtgt 20 <210> 134 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 134 agggcagcct tgtctccaa 19 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 135 cctgccagat tctcaggctc 20 <210> 136 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 136 catcacaggg gcacagactg 20 <210> 137 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 137 gatttgctga gggcagggt 19 <210> 138 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 138 ccccaagtct gatccttcct t 21 <210> 139 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 139 gctgaccaac gatcgcctaa 20 <210> 140 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 140 taagcgccac actgcacct 19 <210> 141 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 141 cctgccagat tctcaggctc cctg 24 <210> 142 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 142 ctgattggag acaaggctgc cct 23 <210> 143 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 143 ccttcatact cttgcatcct cccttctcca 30 <210> 144 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 144 ttccttctct tctgtgactc aattatttgt ggaca 35 <210> 145 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 145 tctggcacct cagcctccct ggccatcact gggctccagg ctgaggatga ggctgattat 60 tactgccagt cctatgacag cagcctgagt ggttctgtgt tcggaggagg cacccggctg 120 accgccctcg gggctgatgc tgcaccaact gtatccatc 159 <210> 146 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 146 tctggcacct cagcctccct ggccatcact gggctccagg ctgaggatga ggctgattat 60 tactgccagt cctatgacag cagcctgagt ggttatgtct tcggaactgg gaccaaggtc 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159 <210> 147 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 147 tctggcacct cagcctccct ggccatcagt gggctccagt ctgaggatga ggctgattat 60 tactgtgcag catgggatga cagcctgaat ggtgctgtgt tcggaggagg cacccagctg 120 accgccctcg gggctgatgc tgcaccaact gtatccatc 159 <210> 148 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 148 tctggcacct cagcctccct ggccatcagt gggctccggt ccgaggatga ggctgattat 60 tactgtgcag catgggatga cagcctgagt ggtcgggtgt tcggcggagg gaccaagctg 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159 <210> 149 <211> 153 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 149 tcggggaaca cggccaccct gaccatcagc agagcccaag ccggggatga ggctgactat 60 tactgtcagg tgtgggacag cagcactgct gtgttcggag gaggcaccca gctgaccgcc 120 ctcggggctg atgctgcacc aactgtatcc atc 153 <210> 150 <211> 156 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 150 tcagggacaa tggccacctt gactatcagt ggggcccagg tggaggatga agctgactac 60 tactgttact caacagacag cagtggtaat gctgtgttcg gaggaggcac ccagctgacc 120 gccctcgggg ctgatgctgc accaactgta tccatc 156 <210> 151 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 151 tcagggacaa tggccacctt gactatcagt ggggcccagg tggaggatga agctgactac 60 tactgttact caacagacag cagtggtaat catagggtgt tcggcggagg gaccaagctg 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159 <210> 152 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 152 tctggcacct cagcctccct ggccatcact gggctccagg ctgaggatga ggctgattat 60 tactgccagt cctatgacag cagcctgagt ggttatgtct tcggaactgg gaccaaggtc 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159 <210> 153 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 153 gatgcttcgg ccaatgcagg gattttactc atctctgggc tccagtctga ggatgaggct 60 gactattact gtatgatttg gcacagcagc gctgtggtat tcggcggagg gaccaagctg 120 accgtcctag gggctgatgc tgcaccaact gtatccatc 159 <210> 154 <211> 153 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 154 cttgggggca aagctgccct gacactgtca ggtgtgcagc ctgaggacga ggctgagtat 60 tactgcctgc tctactatgg tggtgctcgg gtgttcggcg gagggaccaa gctgaccgtc 120 ctaggggctg atgctgcacc aactgtatcc atc 153 <210> 155 <211> 153 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 155 cttgggggca aagctgccct gaccctttcg ggtgcgcagc ctgaggatga ggctgagtat 60 tactgcttgc tctcctatag tggtgctcga gtattcggcg gagggaccaa gctgaccgtc 120 ctaggggctg atgctgcacc aactgtatcc atc 153 <210> 156 <211> 165 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 156 tcaggcctga atcggtacct gaccatcaag aacatccagg aagaggatga gagtgactac 60 cactgtgggg cagaccatgg cagtgggagc aacttcgtgt ctgtgttcgg aggaggcacc 120 cagctgaccg ccctcggggc tgatgctgca ccaactgtat ccatc 165 <210> 157 <211> 164 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 157 tctggcacgt cagccaccct gggcatcacc ggactccaga ctggggacga ggccgattat 60 tactgcggaa catgggatag cagcctgagt gctggccccg ggtgttcggc ggagggacca 120 agctgaccgt cctaggggct gatgctgcac caactgtatc catc 164 <210> 158 <211> 22800 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 158 aagctctaaa actacaaact gctgaaagat ctaatgacta ggacagccta gtaattttca 60 taggggcata aatgtgaaac gccttgtgca tcgtagaaga aagcagaaga gaaagcattc 120 ccaatttctt aactgccttt tacctatatt aatcagtaat atactggctt ttacctctgt 180 taatcataat aaacaaattc tcaataaatt ttatcgatac tcttcaatgc ctgctcagca 240 acattttccg aaggcagctc aagatattaa ataactcata agggccaacc tcctattgca 300 gcattctttg ggatttaacc agtttcccaa gactcttttc acaatgttaa gatgttagaa 360 atagatccaa aactaggtga tatatcccct agtaaaactg tgaggtcaaa cttgtctggc 420 taatgcttcc atttaaaaat ttctctttct tgatccttca ttgtatgtac acaataaatc 480 aggggaaaac tttaactgag tgaatcaaag tattctcatt attataatag gagcttcaca 540 cacacacaaa aaaatcaatt ctattactct cagcctcagt tcctaaagcc aagttaaagt 600 cctgttctaa gatcattgtt gcatgaccat atgtattcca ggtctaatct aaactgtgga 660 taaatcccag caggacatta gagatttttg tgagagtaag catataggat tcagggttta 720 tgagctttag atttttcttg tcaaaatgaa tgagagttgc catatctaaa aattattccc 780 agataaataa aattcactac ctagaattaa tttatgcata taagtagaaa tgctatctcc 840 ctttttacca tccaaagtgg aaagcctcat ggaactagaa attaatatta gaaaaatcag 900 ttaataaaag tatgtcattt catcaattca ataagttata atagcaaaaa accataataa 960 attatcactt aaatgtcaat acatttataa actatggtac ataaatagga tattgaatag 1020 ccattgatgc tcctgatgaa aattagcagg cagtgataaa tgataaatat gaagcacatg 1080 tcaataaata aaataagttt tatgtaattt aggagaaaat ggtgataatg acacaaaatg 1140 tgaattatgg atgcatctat aaaattcttt gtacatttgt gaattgtaaa tatttatctt 1200 agagacatta ttactttgta tatgttccat ttgctcacct atatgtccca gtctccttac 1260 aaatgctatg gccaaagaaa taggcataca tacatccttt gcaggctgag gcaggaaaaa 1320 gatcttacgg aattttccag tctatccttt atctgtataa gcaacttaag aggccatgtg 1380 ctccaaatgg tgcaaataca agatggtaga gcctctgtct gcctggatcc ttgagtggct 1440 gcatggagca gagcaccttt ctggccctgg tgaagattgt agcatgagca agatataagc 1500 atttgttgga gctaggccat gagatttggg gcagtggtat aacctaccct attatggaaa 1560 atataaatac acaaaacaga aaagagagag agaagtgaga gaagactgtg agagaagtgc 1620 atgagagaag actgtgtttt gttcatttcc tataatccta tatcaccatg ggatcctgtg 1680 ccttctggtg atcaaactaa tgttctacag ctccaaagaa gaatgctcgc ctaacgtctc 1740 cattccaatg acctagagac taaaagccaa aaagaacctt agaaattatc tattgcattc 1800 tttgatgtaa ggaaatatct tagagggcac agatagaaat atcttaaccc aggtcactta 1860 gttcgtggca gagctgaggc taaaaccagg ccttttgact cctaattttg tgctctttac 1920 accttctcac atcacttctc caacccaaag tctagcagaa aaggctaaaa taagatatat 1980 gcatagattt gctattataa gtccatgtac ttcctcagac gctttaagat ggggcttctc 2040 atggttcaca ataagcagca gagggaagtg aataactatc ttcgtctccc ctactgctat 2100 ttgtgcagtt tgaagcttat ctcttaaatc atgttttctt ctcgtagtaa atactacaac 2160 ttgtgccttt tatgtgtgta taaattttaa tataattttt ttccatgaac cattcaagta 2220 aaatggacac tccaaaaaga tgttcaataa ggttacatgg cttcacattg ccccctctac 2280 accatcttgt ggagctacac attcacctca cccaaatttg agaaaaataa tcaagaaaat 2340 gactctcact agcagtgaga ccaagtccat aagcactaat gtcatcagtg cacactgcag 2400 cctcatgctg ccaagcatgt tttgggcgta tccctggact ggtttggtga catgatcaaa 2460 ggtacatttt ccacctgcat agccccatcc tggatctata gccttccttg tgtctttgtg 2520 aacaacctag tgtgaactca aagtatgaga cagatctcaa ttaatttaga aagtttattt 2580 tcccaagatt aaggacaagc ccatgataaa gcctccagag gtcctgatat atgtgcccaa 2640 gggggtcggg gcacagcttg gtgttataca ttttagggag acaagaaaca tcaatcgata 2700 tgtagaagat gtgcatcgct ttggtctgga aaggtgtgac aactcaaggc agggaagggg 2760 gcttcctgct ggggttgcat tgttttgagt ctctgatcag cctttcacat gtgaaaggca 2820 ggtagagaaa tagtcattta tgccttagtc tggcttattg aaacagtagg gcagaagaag 2880 cattgcatat gcatttgtct gaagtgaaca gagggatgac tttgagctct gtcctttctt 2940 tgtccacaag gaattacctt gtgggcaaat tgtgagggag gtatgtagct tttttttctt 3000 tgtagctatc ttatttagga ataaaatggg aggcaggttt gcctgatgca attcccagct 3060 tgactttccc ttttggctta gtgatttttg gggtcctgag gtttattttt tctttcacat 3120 tagtataact acttttcttt ttctaattcc ttttctactt gtatgtgtta cagctgactt 3180 atgttacttg caaaaagaat tctgactaat gcaccatctg actagaaggc agggttcttc 3240 gatgataacg aatcctccag aatctagtaa acagaattgc ctgaaaaaga ggtgggtgtc 3300 ttcttgggga atttctcatg gcaatgaatg gcaactggcc aaaggattta tgaccagact 3360 gagctctctt ttatctattc tgttactcac caagacctat tagggtttgt gctccacagg 3420 gacactggtt tctaagttct agggttaaac agtccactcc caggcccacc acaccatacc 3480 ctcctgacat ctggtgaaca gcaataaaat tgtttcttat tctgaaaatc ctccaatact 3540 tccaccatcc ccaaaaatgc agtggaggag gagagaaaat gaattgttcc attagagaac 3600 acaatatcca ttatattatt cttggccttt gagatacctt acaaaacaaa tacaaaaaaa 3660 gtcccaattt aacatctttt aataatcttt acaaaacaga acacatctcc tttcttgata 3720 atagtcaaga ggctcagtgg caactgtggt gaaaagtgtc agattctggt catgtttcaa 3780 aggtagaaaa aatagaattt gttaacatat tggatgtgag gcgtgggaga aacgtgaaat 3840 caaggtggtt gcaagtgttt aacctgagca actagagaat ttggaaggac attttctgag 3900 atggggaagg caggcgggaa tcagggatta gagttgaaca tattagacat ttgagatgcc 3960 tgctagacct ctaattggca atatcccttg gacaggtgga tgaatatgcg tgattctgga 4020 gttcgggaaa tagtccgggt ggagatgcaa atttgggaaa cagggcgagg ttactagcaa 4080 tgagttaaat caatgaaggc aggctgggac ctggcaggta acccaacaag tagaggtcga 4140 agagatgaga agaaaacagc acaggagact tagaagcagt ggtcaggagg aaggagttga 4200 accaagaaag tgatgtccca gagccaacaa aataaggatt tcttttctgt ttacaaatgt 4260 aaaattaaaa ggtttaataa aaagaaaatt tacttttatg gttggttgtt attaagtggt 4320 ccaaacactg tctcctattt gtagaatcag aactctctca tggcagtaga aaatttggaa 4380 agttactttt taaaaggtgt gtgcactgct gccctttgct ggtcaagttt atgcactgca 4440 aattccaagg acgattgctc gtcagctttt ctcctttaaa atagctcagg ctgtacaagc 4500 tagaaagaac ctcgcaagat attccttcca acatttgcat ttgacttatg ggaagtgcag 4560 gttcagccag aaaagttgtg tgcaaggccg tttatgtaag tttatcagac ctgattctta 4620 cggctcttcc cattgtttcg agcctccctt ccattcactt cccgctcata cgcgaccaag 4680 tataggacag gagtagttat tctgcacttt atagcagctc cactgtctgg cactctgatg 4740 ttctttaatt acaagcttta tgacagtgat tctcaacctg ctccactgcc tccacctagt 4800 ggcagaaaga agaaaatgtg tgtaactcgg gagtctctgg tctgaaagct ccggggtatc 4860 atttcttcaa agtcttgagc ttgtttttgt ttgtatttat ttatttattt gttttagaga 4920 caaggtctcg cactgcactc cagcctggga gacagagcga gacaattcag gatctatcta 4980 gtgaataaag agatatcagt aatgactgtt ttatattgtg gctgtagcgc attcgaggga 5040 taattcgatt ctgttctgct ttcgaatgca tggctcactg taacctccaa ctcccgggct 5100 caagcgatcc tcctacctca gcttctccag tagttgagct tgatttattt taaagtttca 5160 taaaattttg gcatttcttt ccacaatatg gccatgtgtg ctttactata aaatattttc 5220 atcacaaaat ttacatcgct ggaaatcccc ataagccagt ttgagaaaca caacccaaga 5280 aagcagaaca gactcaaatt atcccttaaa tcccccttaa ccacaaatat aaaacagtcc 5340 gtgactgggc gtgttggctt acacctgtaa tcccagcact ttgggaggcc aaggcgggtg 5400 gattacttga gctcaggagt tcaagaccag cctggccaac atggtgaaac cccgtcccta 5460 ttaaaaatac aaaattattc aggagttgtg gcaggcagtt gtaatcccag ctacttggga 5520 ggctgaggca ggagaatcac ttgaacccag gaggtggagg ttgtagtgag ccaagattgt 5580 gccagtgcac tccagcctgg gcaacagagc gagacttcca tcttaaaaaa aaaaaattaa 5640 gtaaataaaa tataaaaaaa taaagcagtc cctattgata tctctttatt cactaaatca 5700 acctggaatt gacctgaatt ctgatttttt tttcatcatg gattttttgc attaattttg 5760 attgtttaaa tattgcatta aaatattatt tatcttgact actgagtttg cgggacctcc 5820 ttaaaattta tgaccaaggc aatgcctcac tcactcgcct taccataatc tgggccacat 5880 atcaggggct ccaatagcaa gcaacatgac ttttgaacag ctaagacttc tctcttcact 5940 gtgaagacca gatgggccct gcaaacagtg taacctctac atgaaaatgc acgagattcc 6000 aactacaacc aggcacaaaa gactctgatg gtgaagtccc agccctccaa gtcccaactt 6060 cctgaaggga aagagcaccc caagttctga ccagaggcca gagtcataac gaagatggaa 6120 tgtgagcttg acatagaagg ggtggtagca cctggctcag taatgaagag gctttcggtc 6180 ctgaaggaag agctcagcac attcaaagat tagaagggag gtcccagtca taggagcagg 6240 gaaggagaga aggcccaata agaaacacag acaggaggga ggggtcaggg caagatcata 6300 ctggaaacaa ctagagagct aataaaagtc acagtgccca gtccccacat ggaccagact 6360 cttcggaatc tctaggcatc aatttgggca ccagtagttt tcaaagttct ccagaagatt 6420 ctatgcacac cagccaaggg tgggaaccac aggtgttggc ctagggatca tgacaatgag 6480 tttctaagtg caataagaaa cctccagaga gtttaagcag gggaataatt tgatttgttt 6540 cttgtttgtg atttttaaag atcagtctgg ttactgtgtg taagacaata atccagaaaa 6600 tctgttgctc atgaaccaca tatctgtaaa tttgcttccc ctgtaactgg atctaaccaa 6660 caaaaattag tacttactaa gaaattacat gcccagggac tatgctaagt aattcataaa 6720 cactatttta tttactcctc acagcaagtt tataagagaa acgttattat ttccacattt 6780 cggatgagaa atttgaggct tggggaaagt taagtaattt acctaatgtc acacccagtt 6840 cataagatgc agagttaaga ttctaattct gtgtctaagt tgatgctcca tcaaacacac 6900 cacgcctcca actaggaagc aacatgctgg ccagaggatg ctgtcatcaa gtttacagaa 6960 tggttagatt tctaggcaca gatgaataaa tcaacatgtt ggtttgcaat agaatgaatc 7020 tatccagctc tgaatttgca tccaagggtt tgtgagcaca caagtctaaa agtgtggcct 7080 cagctctgct aacttcatca aggtgaatac ctaggaggcc accctctgag accaccagat 7140 ggacagtcca ccatctgttt acagatggta aagccacata ccagctttgc catctgatgt 7200 tctctattca cattcaacat ttatacaaga aatagtcata tggatccttt tcaatagaca 7260 gtactgggga aattgaattg ccatatgcag aagaatggaa ctagacctct atctctcacc 7320 aaatacaaaa gttaactcaa gacagattaa agacttacat ataagacctg taactacaaa 7380 aacactagaa gaaaacctag ggaaaatgct tctggaatta atctaggtga agaactcagg 7440 actaagatat caaaagcaca agcaccaaaa caaaaataga caaacaggac ttaattaaac 7500 tagaacgctt ctgaacagca agagaaataa tcaatagagt gaacagataa tctgcagaat 7560 gggtgaaaat atttgcaaac tatgcatcct acagggaaat aatgtccaga atttagaagg 7620 aactcaaaca attcaacaac aacagcaaaa taaccccacc aaaaaagtgg gcaaaggaca 7680 tgaatagaca tttttcaaaa gaaggtatat gatatggttt ggctctgtgt ctccacccag 7740 atctcacctt aaattgtaat aatccccaca tatcatggga gagacccggt gggaggtaat 7800 tgaatcatgg gggcaggttt gtcccatgct gttctcatga tactgaataa gtcctatgag 7860 atctgatgat tttataaagg ggagttcccc tgcacacact ctcttgcctg cctccatgta 7920 atatgtgcct ttgcttctcc tttgccttct gccatgattg tgaggcctct ccagccatat 7980 ggaactgagt caattaaacc actttttctt tgtaaattac ccaatcttgg gtatgtcttt 8040 attagcagca taagaacaga ctaatacagt gtacaaatgg ccaagaagcg tacaaaaaac 8100 aaaatgctca aatcactaat cactagagaa tcgcaagtta aaaccacaat gagatattat 8160 cttacagcag tcagaatgcc tattattaaa acaccaaaaa ataacatgtt ggcaaggatg 8220 cagagaaaag ggaatactta cacattatta gtgggaatgt aaactagtac agcttctgtg 8280 gaaaacacta tggagatttc tcaaagaact agaaatagaa ctaccatgtg gttcagcaat 8340 accacaactg ggtatctacc caaagggaaa taaattatta tataaaaaag atatctgcac 8400 tcacttgttt attgcagcac tattcacaat agcaaagata tggaatcaac ccaagtgtcc 8460 atcaacagat gattggataa agaaaacgtg gtgtgtgtgt gtgtgtgtgt gtgtgtgtat 8520 acacatacca caatgaaata ctattcagct ataaagaaaa gaatgaaatc atgtcttttg 8580 cagcaatgtg gttggaactg gaggccatta tcttaagtgg ataattcaaa aacagaaggt 8640 caaatgtcac atgttctcac ttataagtgg gagctaaatg atgtgtacac atggacatag 8700 agtgtggtat gataaacact ggagattgag atgggtggaa gggtggaagg aggttgagtg 8760 atgagaaaat actaaatgga tacaatatac atgattcagg cgatagatac actaaaagcc 8820 cagacttcac cactacacag tatagctatg tagcaaaatt gcacctgtat tgcttaaatt 8880 tatacaagta aaaaaaagat cgtacgaatt ctgtttttta ttctctatga aattactact 8940 gagagtatta tccaatgccg tttctatgca gtgcccccaa tattatccat ttagcagctc 9000 ctatgcaatg ccccaagata gaaattgtct tcaactttta tcccaggaaa accttcagtc 9060 acacgtagaa actagaaatt tttcccctag atgaaagtta tgtaacataa cacattatct 9120 tcatttagtc ggtttccaag aagctcagaa ccagatttta tgttcaatca aaaactgctt 9180 attttaagtg aggtttactg aggtataaat tacaataaaa gccacctttt cgtgtatatt 9240 tctataagtt ttggcaaatg catagctgtg taaccacaac cacattcaag atataggaca 9300 agtccctcat cctttaaagt tcctttatgc cccttccttc accccagccc ttggcaacca 9360 ctggtttttg tctgatccaa tcgtttgcct cttcctgaat gtcatgtaaa tagagccatg 9420 caatgtgaag ccttttgagt ctggctttgt tcacttgttc acttaggaga atgcatttga 9480 gattcatctt tgctgtttcg tgtagcacta gttcactgtc tattgttgag tagtattcca 9540 ttgtgtggat atgccacaga ttgtttatct agttaacaat ttaaagccat ttggtcattt 9600 ctaattttta gctgctaaga ataaagttgc tgtaagcttt ccaatgcagg tttttgtgtg 9660 aactcaggat ttcatttcgc ttgggtaaat tcctagcttt gggactgctg agtcatctgg 9720 taggtgtatg ttgaacttta taagaaactg ccaaactgtt ttccaaagtt gctgtgctct 9780 tttgcactcc catcagcagt gaatgagggt tccacttgct cgagcctagt attttaactt 9840 cactatatac cttctttgat gacatatcct ttcaaatttt tggtcaagtt tttattgggg 9900 tgttgttact atggactgtg agagttcttt gtatattctg catatgattt ttttctcaca 9960 tttgtgtttt atgaatatgt tctcccaatg tgtggcgcct tttattttct taacgtgcca 10020 tgtgaagagc agaagtttaa ttttatgatg tccaaattat ctttttttct tttctttttt 10080 agatcaaaat aggggtctat tttgattacc actgttattt tatctccatt tgattttcga 10140 tttttatttt tatttttcta atttcattgt aaatttttaa ttaaacccaa atattctagg 10200 ggaaagaggc aagataaaaa tagtctaact tgggcataaa ttttagagtc atattctctt 10260 gccgagaaag gaaactagct ctcttacatt gattgtttaa tttcagacgt cactacttta 10320 tgaggatgcc caaattatgg gctttaaaaa atatatatcc aaacaggggt tcagaaagaa 10380 taactaattt gtccacaaca acacaaaaaa tgattccacc ataagtttgc ccagtgacag 10440 ggtctatatt attttctata tatcaaattc tacaactggt tcttaaagct actgtacata 10500 acctaagtta aaatattagg tattagttga taagacattt tatcatctat gaaatgttgc 10560 ctgttgtcat agttagagaa tcttttaaaa tatggagcta ttttcataga ttaaactatg 10620 ccagttaaaa gttgggtaaa aagaactaca gaataatatt tatgtttatc gtgtaaggtt 10680 ttaaagcaaa ctccaagtca ttttcatcaa tgaaatcaat aaggttttgc aaatatatat 10740 gtatgaaaat actgatttaa aatgcaaata aggggagagt ttgagagaga gagagagacc 10800 aaatgatttt ataattctag taagtttata ggtttatggg gtttttacgt acttttctac 10860 ccaacttgtc tataagactt taatgaatca cttagaattt ttaaaataat ttattattac 10920 tctgtacctg ttctttactc tgcaaatctt accttgccct tttgtctaaa agcaataaaa 10980 tctgacctgg tttatatcgt atcattgatt ttgttactta gcaagcacag tgatccatta 11040 ggcctatgta ggctcatggt ttatacaaca ctgccatctg ctgacagagt gtgacagtca 11100 cagtcagcaa cacgagacca ctttattttc atttttagtg tttatagaaa tatgaatata 11160 cacaaatagt ataatgaacc ctaagcttca caaattaaca ttttgctaat cttgtttcaa 11220 ctaccgcctc ccccctcatc caattactct gttctctcac ctcctcacac acagacactg 11280 gcagtatttt tcagccaatc attaatacgt tgccaactga taaggacttt taaaaaacaa 11340 ccaccattcc attatgattc ccagcataat tgagagtaat tccctaatat ccaataccca 11400 ttttctattc caatttcctt gattgtcttt aaactgtttt taccctaagt ttgcttaaat 11460 caaagtccag gtcctgttaa acatatggtt aagttttacc caaacccaaa taaataaata 11520 aataaataaa taaataacct attttttcca attccaggga atagtgaaag agggtaaatg 11580 ccattattta gaaacataaa tcacatcata ggactagaat tatcttgaag tcaaaattga 11640 agactgaaaa tggaaaagaa aggtatagac taaacttatt taaaaacttc aatgcagaac 11700 tctaagagaa gatattagaa agttgtacca gcattcatta ttcagtattc atcagtattc 11760 actcagctat atgtagttga aatctaacta gaggagcttg atcagataaa gagatacatt 11820 tttctcacca aggcggactc tggaggcagg tggttcagag ctagacagct gctgcaggac 11880 ccaggtcctt tccctgcctg ctcctccact ctagcttgtg actttcatcc tgcaagatgg 11940 gtgtttctgc caagttccag atagaagaag atagaacaca aaggagaaat aagcagtggt 12000 gcctctgtcc atcaagcaaa atttttccag aaatgcacaa tagatttcag atgatgtctc 12060 aacagtccta actgcaaaga agctgaggaa ttagattttt ggctgggaca ctgttgccct 12120 gtaaaaaaat tgggattctg ttattaaaga ataagaggag ggaagaaaga ttgaaaactc 12180 ctatgcaata gtgaaaaaaa taagaaactc aataaaaaag tgggcatacc ttaaaaacag 12240 gcaattcaca acagatgaga ccccaatagc caataaacat ttttaaatgg tcaacctcat 12300 gagtgatcag aaaacacaaa tatgtatttt aaaccaaaaa taaaatacaa tgtattgacc 12360 atttgagtgg aaaaaaatta aaaagcctga taatatcaag tattggagag gatgtagagt 12420 gaggaaactc catggaggac ctatcattgc aaatgtggga atgaaactta atacacgaat 12480 ttgaggccaa tttgtaaatt gaaaaatgcg cacaccctgc aaccaagtac cccttgcaat 12540 atttttgaaa agacaaaaac gttatgtaaa tggaatcatg caatatgtga cctttatact 12600 cagcataatg cccctcagat ccattgaagt catgtgtatc aacagctcac tatttttttt 12660 ttaatttttt ttagagacag agtctcactc tgtcacacag ggtggagtgc agtggcgaga 12720 tcataactct ctctagcagc ctcgaactcc tgggctcaag catcctcctg cctcagcctc 12780 ccaagtagct aggactacag gcatgggaca caacacacag ctaatttttt taaatttttt 12840 ttagagacat ggtctcacta tgttgcctac gctggtctca aactcctagg tcaagcgatt 12900 ctcccacctc tacttcacaa agtgctgtag gtatgtaggt atggattgta ggtatgaacc 12960 accgtgccca actcactact ttttattact aattattcca tgggatggat gtaccgcagt 13020 ttgttttacc attaatctat tgtaggacat tttgactgat tccagttttt ttttaataca 13080 aataaaacca ctatgaatag ttgtgtattg tatacgtttt tgtgctaagt tttcattttt 13140 ctgggataag ttttcatttc tttgggcttt tactgtatcc ttgatattat aatatgttac 13200 atcttcagtt ttattctatt caatatataa tcttttattt tccttgaaat ctcccatgga 13260 ttgtttagaa gtgtgttgtt ttgtttccaa gggtttggca tttttcccat tatttttcta 13320 ttatcgattt ccagtttgat tccaggtggt cagagaacac acttcatgtg atttcagttc 13380 tattaaattt gttgaggttt gttacatggc ccagtatatg gcaattttgg tatatgttcc 13440 atgagcactt gaaaagaatg cgaattctgc tggtgctggt tggagttttc cagcaatgtt 13500 gatttatgat cttactcatt gatggtggtg ttgagtttga tgtgttctta cgatggcagc 13560 tttaacattc ttgtcaggta attctaacgt ctctgtcatg tcagtattag cgcctcttaa 13620 ctgtctcatc aaagctgaga ttttcctggt tcccctggtt cctgttggga tgtgtggttt 13680 tcatttgaaa tctggacttt ggagtattgt gttatgaggc tttggatctc atttaaactc 13740 atctcagcga atttcctctc ttgccactca ggaaggagaa gttgggtgtt tgaatggagc 13800 agagccgtta ctgcctaaga attgttttac tgggcttccc ctttctttct cctttgacta 13860 gagagagcca gctttttatt agggctttat gtttttctgg gcctgttggt gtttctgggt 13920 tgacaaactt ctccagaacc aagtctggaa tggatgaggc aaaaagaaac cccgtggaat 13980 gcactgctgg gtcgctcctt gggtcccaat gttcctaact ggtctgcctt cttctctcca 14040 gcttccagag tcttcataag tttgctttac gtacaatgtc cggggttttt actttacttg 14100 agagaaatag gtaaaagtaa ttctactcca tctttcagga agcaaaagcc cccttgtgta 14160 tttttttaaa ctttcaaaaa caaaacaaaa ggcagctgca acagtaaaga agctagtaac 14220 acccttggtg ggaaattcaa gtccaaatac acattttaag tttggctagc cagtgagaac 14280 atcagaatag ttcaggtttt aaacaaattt atatttatga ttatgcatat actaaaagct 14340 gaaggcatct tatatttact aagcacctat tttgttcttg ttaaaaagac agaattccat 14400 tccctaggaa atttgacctg gcagctggag ctgatccacc tggccactag agcacagagc 14460 agggagagta gtagccctgc cccagccacc cctcaagaca ggattctttc tctgggaact 14520 gtaggtaaca ctaaatcgtt ctggaacaca acaacgaaag aagaaaggaa agagaaagaa 14580 agaaaggaag aaagagagag agaaggaagg aagggaggga gggaaggaag gaaggggaag 14640 ggaagggaat ggaagggaag gaaggaagga aaaggaagga agggagggag agagggagga 14700 aggaaggaaa ggaaaggaag gaaggaagaa ggaaagaaaa aaagaaagaa agaagaaaga 14760 aagaaagaca agaaagaaag aaagaaagaa agaaagggga aagaaaagaa agaggaaaga 14820 aagagaaaga aagaaaagaa agaaaggaaa gaaagagaaa gaaagaaaaa gaaagagaaa 14880 gaaagagaaa gacaagaaag aaaaaggaaa gaaaagaaag agaaagaaaa gaaagaaagg 14940 aaagaaagag aaagaaagaa aaagaaagaa agaaaagaag aaagagaaag aaagaaagaa 15000 aaagaaagaa agaaagaaag aaagaaaaag aaaaagaaag gagaaaatga cagcaattac 15060 ttttgcaaca acctaatata agttttttaa aagttaaata ttctgttcca tgcattgctg 15120 gataccttat aaataacagg gcatcctatg acctgaattt cccaaattat gagttgaggg 15180 tttgaactag ttttaaaaaa caaggaggcc aggcgcactg gctcatgcct gtaatcccag 15240 cactttggga ggctgaggca ggtggatcac gaggtcagga gctcgagacc agccttacca 15300 acatagtgaa acaccgcctc tactaaaaat acaaaaatta gccgggcgtg atggtgcgca 15360 cctgtaatct cagctactca gcaggctgag gcaggagaat cgcttgaacc cagaaggcgg 15420 aggttgcagt gagccaagat cacagcattg cactccagcc tgggcgacag agggagactc 15480 cgtcttcaaa aaaaaaaaaa aagacaagga atctgtaaaa caggcactgg aagtatatgc 15540 acttttattt tcattctatg ctatccgatg cctactgcta tttcccttca tatttaacct 15600 ccaacagctg cattttgctc cctccagacc acctgattgg agctcacgtg ctcccacaca 15660 gtacctccaa ccagagagag tcgagtccca cagaaaggcg taacaatcac cagtaatttt 15720 gcacttattt tacattgtgc cttgatacag agtactcaat gaatgctctt tgaatcatat 15780 ttaataaata tgtgtatttg ggattgtagc atattgcagc tacctggata tataatttaa 15840 ttagaaaaaa aattttgtgt ggctcaatca acaaacgact tttctctctc tctctttctc 15900 tttctccctc tctctctctt tcttctcagt tgatgttgct ggagttcagt gttgtgcaga 15960 tggcagtgac aaatgccatg ggcacatgag atatgataaa aggtccctga agaaggtgga 16020 gaaccagtta tcttatgaaa ttttccagag tgggtactgg atctctcctg tctggcacca 16080 tgctggcctc agcccaaggg gaatttcctt ccagagacag agggcagtga ttgaggtggg 16140 gagacagatc gtaacactga gacttacatg aggacaccaa acagaaaaaa ggtggcaagt 16200 atagaaaatt ctttcttctg gacagtcttc tctgttctaa cttcagcaaa attctccccc 16260 cagtggatgc tattgcacaa ccctacatat gctatgtttt ttcctataca cacttaccta 16320 tgataaaatg cattaattag tcacagtaag aggttaacaa caataactag taataaaata 16380 gaacaattca gtaaaataag agttacttga gcacaaacac taggatatca tgacagtcaa 16440 tctgatgacc aagagggcta ctaagcatct aaacaggagg gtaagtgtag acagcatgga 16500 gacgctggac aaagggatga ttcagtccca ggctggtatg gagcggaagg gcatgatatg 16560 tcatcacgct actaaggcac acaatttaaa atgagtaaat tcttatttct agaaatttct 16620 ttttaatatt ttcagactac agttgcctac aggtaactga aaccccagaa agcaaaattg 16680 ttgataagga ggtactactg tacatcgtcc tttgaaccaa ctttatcatt tgctagtata 16740 tacatatata cctacataca tacatataca catacctgca cacacctata tgtatacgta 16800 cacacacaca cacgcacaca cacacactca catctactaa tgttagaata agtttgctaa 16860 ataagatgca caacttgtta atgtcctaca gagcaataaa accataagca ttggggttat 16920 cttttctact agataaaaat ccattatcat tttcataaag ttttctttac attaacatct 16980 aacttttgca atctagtttt taatcatcat aaataggaag caaatgaact gtttctctag 17040 tgaatcaaat atccttgaaa acatacatag tcatcttttt ggtttatttt tatttttaga 17100 taaattattt aaagttttaa ataatttaac attcacaata gtttgtgact gtatattttg 17160 acttggtcct tcaaacttaa tttgtacttt tatgtatcgt gcttacctca attttttatt 17220 cacttttcct aaactttgct ggattggttt attatttttg tctatttctt ttccttctag 17280 tggtttggga gggtttttta aatcccatta ctattgaatg cctattaact tgcccccttt 17340 ttctttcaat ctctattccc acggcctgaa gcatgagggc caagctgtct gtaaccagca 17400 gagagatgac ccaggtgtta ttccactctc cactgtccac ctatcaccat tcccagcccg 17460 atagctctga agtacggctt ttctggggct ctgtggggaa aactagaact ggctgcttca 17520 aggacacctc ctgtttttgc aatggaaaaa atgtttctaa attccagttt ctctatgaat 17580 tcaatgacat ggtttaaatc tctgtggtgt tcttcaaagt tttttcttct aataggacct 17640 ctcatgattc tccaaccacg aaataaattc attatcattt ttatatttct tctgtcattg 17700 caaaggaggt tttgaaagag tggaggacgc gctaatgaac tcaaaaatcc acactattcc 17760 ttgtttccat ctgttgttca ttcattgttt ccattggcct gtccgcctcc tatcctcctt 17820 cttagacttg gagctctagc ctcagccagg atagggaaaa gagagatcag actgttactt 17880 tgtctatgta gaaaaggaag acataagaaa ctccattttg atctgtatcc tgaacaattg 17940 ttttgccttg agatgctgtt aatctgtaac tttagcccca accttgtgct cacagaaaca 18000 tgtgttgtat ggaatcaaga tttaagggat ctagggctgt gcagaatgtg ccttgttaac 18060 aacatgttta caggcagtat gcttggtaaa agtcatcgcc attctccatt ctcgattaac 18120 taggggcaca gtgcactgcg gaaagccgca gggacctctg cccaggaaaa ctgggtattg 18180 tccaaggttt ctccccactg agacagcctg agatatggcc ttgcgggatg ggaaagatct 18240 gaccgtcccc cagcctgaca cccgtgaagg gtctgcgctg aggaggatta gtaaaagagg 18300 aaggcctctt gcggttgaga taagaggaag ccctctgtct cctgcatgcc cctgggaacg 18360 gcatgtctca gtgtaaaacc tgattgtaca ttcgttctat tctgagatag gagaaaaccg 18420 ctctgtggct ggaggcgaga tatgctggcg gcaatgctgc tctgttgttc tttactacac 18480 tgagatgttt gggtgagaga agcataaatc tggcctacgt gcacatccag gcatagtacc 18540 ttcccttgaa tttacttgtg acacagattc ctttgctcac atgttttctt gctgaccttc 18600 tccccactat caccctgttc tcctgccgca ttccccttgc tgaggtagtg aaaatagtaa 18660 tcaataaata ctgagggaac tcagagaccg gtgccagcgc gggtcctccg tatgctgagt 18720 gacggtccct tgggcccact gttccttctc tatactttgt ctctgtgtct tatttctttt 18780 ctcagtctct cgtcccacct gacgagaaat acccacaggt gtggaggggc tggacacccc 18840 ttcgagccag gattatcagg gcatttgggg gtctgcaaaa ctaagcccca actcatcgat 18900 ttcacaactt catccagagc cagcctgaac agtagttgcc catgatttct atgccttaat 18960 acgagaagag aacatagggg ctgggtgcca agtaggtaga cagggagggc agggaactct 19020 aagacagagc ttgaggggct cattcctctt gcaaaatgaa acaaaaacca cagcactgaa 19080 tatgtaaatc tcggtggctg aacccctcct aggatagtaa gccctgacac aattgctgct 19140 atcttctctt tctctcaagg aagtcaaaaa acacctgcag ccttactgtc cccttggaaa 19200 caagatgaac atctacattt tctaaagtgg gacaagaatc tctgttcata tttatgtccc 19260 atgcatttgc acgtggccgg acaaaggact ttgcttctgc cagcacatct gtcttcagat 19320 atgagaggaa acagacacaa cctggaggcg gcaaagaagc agctctttct caagtgacct 19380 cctctatctc cctacttcct ggctaatggg gcagccttga tccttgggaa tccaggacag 19440 atatccactc gtgacaaact agctggaaga atgacaacca atcaggttcc aagcaccact 19500 ggatgtgaac cacagaattt cctcctctcc ttgtggaatg tcagcttacg tctgacaaaa 19560 aatgtaaaac tgagagagtt acaatcttaa ggaggagtca agctaaagca gaaagaatca 19620 cctactctgg actccagcat gactgctgag ctcaaatata tatagagaga gaaagaacca 19680 caaacttgaa gatggatatc agctacagac tttcctgagt caggtaggga aatggccatc 19740 cctcaaacct tgcaaaaggc aaacttatgc cattgtgtcc tctgacatac tgggtgatgt 19800 actgtatgtt actgatgtga ggggaacttc ctaaattggc tagtaaatta tgccaaataa 19860 aaagcaaaaa tgatatttct tgaaatgtta catctgagga acattgctaa aataatttat 19920 cagtagtttt caggatgatt tatagatgtg cattgaagtg tgtacttgtg ctctctctct 19980 cctctctctc tctctttctc tcctctctct cgctctttct ctccttgccc ccctccctcc 20040 ctgactttcc ttcctgtccc ctccacagca gtttatattt tttttctgat aatctaactt 20100 tgctgagggt tcaatgtaaa gcaccttcag tgatgagtta gttggaatgt tccccaagaa 20160 attctatttc cagcactctt ttacatgaaa tccaagaagc tctcagacta tcttactgac 20220 accttgcctt tcctcaacag atcaatctta tcaatgtcca tcacagatat tttgtagaac 20280 ggtggatcct ggcagagtct cacagatgct tctgagacaa catttgcttt caaaaaatga 20340 accacacaca tcctaaagat ctcagccact tcccatgttt cattttgtgt tacagcaaac 20400 atcacaacaa tcattcctac agatcaccac tgcatgtgat caataaaata gtttttgcaa 20460 caatggtact tatgataatc atcttttatt gtttacaaat actgctttac aatagttatt 20520 cggttgcact gttcatatta gatttccaat tagctcactt aggaacataa gtccctcgaa 20580 cagctcagtc atctttttca ttcctgtttc tatcccctac atctcctcc tttgcagacg 20640 actatctcct acactgaaac aggaaagctt ttaccttttt ggcatgcttg atttaaagat 20700 tatagaaaag tatttgacaa agaaaactca cacatgtgtg tacatatctt ttaaaaagtt 20760 atgtttatgc attgcacagg aatatcgaga atgctaatag gcaatgtcag agtttactgt 20820 ttttcaaaat tagtacagtt ttattatttc taaaaactat aaaatgaata tattcacatc 20880 accatacaga agagtaggag gagatggcat aaagtgtcat tgttcctcct ctgcaatccc 20940 aggagataac taccaagcac aatttatgtc ttttaaaatt cagcccgtat ttatatacat 21000 atatattcaa tgtagatggg atcatgatat ctcaccacac atactcttca gtgacctgca 21060 ttttcacaaa caccttccac gtaactatat agaagtctac gtcttcccct taatgtctgc 21120 tttgtgctac attgtaaagc tctagcacag tttaaccaaa ctcctattaa tgaggatttt 21180 agttattttt cactctttaa acaatatttc catgtgtagt cttatacata cgtctgtaca 21240 cacttatccc agtctaagga gttcctttta ccttccccca tcccagcatt ccctgtcacg 21300 cttgttgctt ccgttgagtg actttactcc tggagtataa tctgcgtata gttcagttaa 21360 aaacatggga tctgagttta ggtcacagct ctgccactta ctgccataag ccagttcctt 21420 gacctctctg ccctcaagtt tttgcaccta caaagtaggg gataatatta gttcctagtt 21480 catagagtct tgggaataat taaatgtgat gatccatgta caatgtctgg cacttagtaa 21540 gtgctcaata aatgtcaccc tttatgattg gtattgcgtg tatgtctgca gagaaaatca 21600 ctttgtgtcc cctttaaaaa aggactatgc ccttggtcag ctattttgca cattaaattt 21660 cacttgccaa tattaactct ccacctctaa cttgatccct ctccttcctc atcttctggt 21720 gagaccaaat gctaattctg ctattcaagg caactagcaa agctgccagt gacagaatca 21780 aataaaccta cccctaatct ttagaattgt agttatgatt tctgttgtaa aagttactgt 21840 tgtggcagtc agtattagtc tttggtctat gatagcatct ctgatctatt attgaytttc 21900 aattakgtat ttttttttat ttattctgaa aatgtttgtt aagcatttgc taagtaaaga 21960 tactggackg agcctcccaa atacagggca aataaaacat caaacagctt ataatttaga 22020 agggtagaag agaatctgaa agcaggtaaa aataaacagg cactcggctg ggcgcggtgg 22080 ctcacgcctg taatcccagc actttgggag gccgaggtgg gcggatcacg aggtcaggag 22140 atcgagacca tcctggctaa cacggtgaaa ccccgtctct actaaaaata caaaaaatta 22200 gcgaggcgtg gtggcgggcg cctttagtcc cagctagtcg ggaggctgag gcaggagaat 22260 ggtgtgaacc cgggaggcgg agcttgcagt gagccaagat cgcaccactg cactccagcc 22320 tgggygacag agcgagactc cgtctcaaaa aaaataaata aataaaataa aaaataatta 22380 ggtactctag gcccagtgac ctgtctctgt actctgtaaa ttcaggtcac ctgctcaggg 22440 ctaatctgag agaaggtctc tcttcagttg aattttgaaa gacaattagc agttcacaag 22500 ctaacccagg tggacaaaga tgttcccaag cagagggagt gcttgtgaaa gctggaggcc 22560 atagaaaaac tctaaggagt gtagggaggt gggagtaatg tatggaaggg gtggagatgg 22620 aaggttaaga gagatacaag gctgcaaaaa tggagctgga ctcaaaagaa aatactgaaa 22680 aggtcttcag tgttgttgat gagattacta tggaaacact atggaacact gggactccat 22740 ggcagctcca aagatggcat gcgcctggtc cagctcagta agagctgagc tcttcctgtg 22800 <210> 159 <211> 154 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 159 tctggcaaca cggcctccct gaccgtctct gggctccagg ctgaggatga ggctgattat 60 tactgcagct catatgcagg cagcaacaat ttaagtcttc ggaactggga ccaaggtcac 120 cgtcctaggt cagcccaagt ccactcccac tctc 154 <210> 160 <211> 156 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 160 tcagggacaa tggccacctt gactatcagt ggggcccagg tggaggatga agctgactac 60 tactgttact caacagacag cagtggtaat cattatgtct tcggaactgg gaccaaggtc 120 accgtcctag gtcagcccaa gtccactccc actctc 156 <210> 161 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 161 tctgggaaca cagccactct gaccatcagc gggacccagg ctatggatga ggctgactat 60 tactgtcagg cgtgggacag cagcactgcc gtcttcggaa ctgggaccaa ggtcaccgtc 120 ctaggtcagc ccaagtccac tcccactctc 150 <210> 162 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 162 aggtggaaac acggtgagag t 21 <210> 163 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 163 ccactcgggg aaaagttgga a 21

Claims (77)

A method of generating a human heavy or human λ light chain variable region sequence encoding a human heavy or human lambda (λ) light chain variable domain, respectively, of an antibody that specifically binds to an antigen,
(a) immunizing a genetically modified mouse with an antigen; And
(b) identifying a human heavy or human λ light chain variable region sequence that specifically binds to the antigen and encodes a human heavy or human λ light chain variable domain, respectively, of an antibody produced by the genetically modified mouse
Including,
The mouse is
(i) (1) insertion of at least one human Vλ gene segment and at least one human Jλ gene segment upstream of the mouse immunoglobulin kappa (κ) light chain constant region gene,
(2) insertion of one or more human V _H gene segments, one or more human D _H gene segments, and one or more human J _H gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, and
(3) insertion of a nucleotide sequence encoding mouse ADAM6 protein, wherein the mouse ADAM6 protein is expressed in the genetically modified mouse and restores or enhances male mouse fertility
Have a gonad genome comprising a;
(ii) generating antibodies upon immunization with the antigen, wherein the antibodies comprise a human heavy chain variable domain operably linked to a mouse heavy chain constant domain and a human λ light chain variable domain operably linked to a mouse κ light chain constant domain; ; And
(iii) a fertility adult,
A method of generating a human heavy or human λ light chain variable region sequence that respectively encodes a human heavy or human lambda (λ) light chain variable domain of an antibody that specifically binds to an antigen. A method of making a nucleotide sequence encoding a fully human heavy chain or a fully human light chain of an antibody that specifically binds to an antigen,
(a) immunizing a genetically modified mouse with an antigen;
(b) identifying a human heavy or human λ light chain variable region sequence that specifically binds to the antigen and encodes a human heavy or human lambda (λ) light chain variable domain, respectively, of an antibody produced by the genetically modified mouse ; And
(c) operably linking said human heavy or human λ light chain variable region sequence to a human heavy or human light chain constant region, respectively, to form a nucleotide sequence encoding a fully human heavy or fully human light chain;
Including,
The mouse is
(i) (1) insertion of at least one human Vλ gene segment and at least one human Jλ gene segment upstream of the mouse immunoglobulin kappa (κ) light chain constant region gene,
(2) insertion of one or more human V _H gene segments, one or more human D _H gene segments, and one or more human J _H gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, and
(3) insertion of a nucleotide sequence encoding mouse ADAM6 protein, wherein the mouse ADAM6 protein is expressed in the genetically modified mouse and restores or enhances male mouse fertility
Have a gonad genome comprising a;
(ii) generating antibodies upon immunization with the antigen, wherein the antibodies each comprise a human heavy chain variable domain operably linked to a mouse heavy chain constant domain and a human λ light chain variable domain operably linked to a mouse κ light chain constant domain and; And
(iii) a fertility adult,
A method of making a nucleotide sequence encoding a fully human heavy chain or a fully human light chain of an antibody that specifically binds an antigen. The method of claim 1, wherein the live genome of the mouse comprises endogenous V _L gene segments and / or endogenous J _L gene segments that cannot be rearranged to form immunoglobulin light chains in the mouse. The method of claim 1 or 2, wherein the insertion of the one or more human Vλ gene segments comprises 12 or more human Vλ gene segments. The method of claim 1 or 2, wherein the insertion of the one or more human Vλ gene segments comprises at least 28 human Vλ gene segments. The method of claim 1 or 2, wherein the insertion of the one or more human Vλ gene segments comprises at least 40 human Vλ gene segments. The method of claim 1 or 2, wherein the nucleotide sequence encoding the mouse ADAM6 protein is located between two human V _H gene segments. The method of claim 1 or 2, wherein the nucleotide sequence encoding the mouse ADAM6 protein is located between the V _H gene segment and the D _H gene segment. The method of claim 1 or 2, wherein the one or more human Jλ gene segments are selected from the group consisting of Jλ1, Jλ2, Jλ3, Jλ7, and combinations thereof. The method of claim 1, wherein the one or more human Jλ gene segments comprise four or more human Jλ gene segments. The method of claim 10, wherein the four or more human Jλ gene segments are at least Jλ1, Jλ2, Jλ3, and Jλ7. The method of claim 1 or 2, wherein all endogenous Vκ gene segments and endogenous Jκ gene segments are replaced with the one or more human Vλ gene segments and the one or more human Jλ gene segments. 3. The method of claim 1, wherein the mouse further comprises a human Vκ-Jκ intergenic region from a human κ light chain locus, wherein the human Vκ-Jκ intergenic region comprises the one or more human Vλ gene segments and one or more genes. Adjacent to the human Jλ gene segment. The method of claim 13, wherein the human Vκ-Jκ intergenic region lies between a human Vλ gene segment and a human Jλ gene segment. The method of claim 13, wherein the human Vκ-Jκ intergenic region comprises SEQ ID NO: 158. The method of claim 1 or 2, wherein the mouse ADAM6 protein is a mouse ADAM6a or ADAM6b protein. 3. The method of claim 1, wherein the one or more human Vλ gene segments comprise Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, or a combination thereof. Including, method. 3. The method of claim 1, wherein the one or more human Vλ gene segments are Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27 or a combination thereof. The method according to claim 1 or 2, wherein the one or more human Vλ gene segments are Vλ1-40, Vλ7-43, Vλ1-44, Vλ5-45, Vλ7-46, Vλ1-47, Vλ9-49, Vλ1-51, Vλ5-52 or a combination thereof. The method of claim 1 or 2, wherein at least two λ light chain enhancers are located downstream of both the at least one human Vλ gene segment and the at least one human Jλ gene segment. The method of claim 1 or 2, wherein the mouse comprises sperm that can migrate through the oviduct from the uterus of a female mouse, as measured by a sperm migration assay. The method of claim 1, wherein the mouse comprises sperm capable of crossing the uterine-tubule junction into the fallopian tube of a female mouse. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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